Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof

ABSTRACT

The present invention is directed to phenylalanine ammonia-lyase (PAL) variants produced by prokaryotes, wherein such prokaryotic PAL variant has a greater phenylalanine-converting activity and/or a reduced immunogenicity as compared to a wild-type PAL. The invention provides compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, and use of such compositions for therapeutic purposes, including the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No 12/421,557, filedApr. 9, 2009, which is a continuation of U.S. Ser. No. 12/107,736, filedApr. 22, 2008 (now U.S. Pat. No. 7,537,923), which claims priority toU.S. Ser. No. 61/066,125, filed Aug. 17, 2007, each of which is hereinincorporated by reference in its entirety.

SEQUENCE LISTING

A CRF copy of the Sequence Listing, titled 11808-069-999_SeqListing.txt,which was saved on Apr. 8, 2009 and is 53,833 bytes in size is submittedherewith and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to prokaryotic phenylalanine ammonia-lyase (PAL)and compositions thereof, and optimization of such compositions toenhance prokaryotic PAL catalytic activity and/or stability, whilereducing immunogenicity and/or proteolytic sensitivity of prokaryoticPAL. The invention further relates to the use of such optimalcompositions of prokaryotic PAL for treating cancer.

BACKGROUND OF THE INVENTION

PAL is a non-mammalian enzyme widely distributed in plants (Koukol, etal., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes7:75-166 (1972); Poppe, et al., Curr. Org. Chem. 7:1297-1315 (2003)),some fungi (Rao, et al., Can. J. Biochem. 4512:1863-1872 (1967); Abell,et al., Methods Enzymol. 142:242-253 (1987)) and bacteria (Bezanson, etal., Can. J. Microbiol. 16:147-151 (1970); Xiang, et al., J. Biol. Chem.277:32505-32509 (2002); Hill, et al., Chem. Commun. 1358-1359 (2003))and can be recombinantly produced in Escherichia coli.

A representative list of PALs includes: Q9ATN7 Agastache rugosa; O93967Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Gianttimber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle)(Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5,Q9XFX6 Citrus clementina×Citrus reticulate; Q42667 Citrus limon (Lemon);Q8H6V9, Q8H6W0 Coffea canephora (Robusta coffee); Q852S1 Daucus carota(Carrot); O23924 Digitalis lanata (Foxglove); O23865 Daucus carota(Carrot); P27991 Glycine max (Soybean); O04058 Helianthus annuus (Commonsunflower); P14166, Q42858 Ipomoea batatas (Sweet potato); Q8GZR8,Q8W2E4 Lactuca sativa (Garden lettuce); O49835, O49836 Lithospermumerythrorhizon; P35511, P26600 Lycopersicon esculentum (Tomato); P35512Malus domestica (Apple) (Malus sylvestris); Q94C45, Q94F89 Manihotesculenta (Cassava) (Manioc); P27990 Medicago sativa (Alfalfa); P25872,P35513, P45733 Nicotiana tabacum (Common tobacco); Q6T1C9 Quercus suber(Cork oak); P14717, P53443, Q7M1Q5, Q84VE0, Q84VE0 Oryza sativa (Rice);P45727 Persea americana (Avocado); Q9AXI5 Pharbitis nil (Violet)(Japanese morning glory); P52777 Pinus taeda (Loblolly pine); Q01861,Q04593 Pisum sativum (Garden pea); P24481, P45728, P45729 Petroselinumcrispum (Parsley) (Petroselinum hortense); Q84LI2Phalaenopsis×Doritaenopsis hybrid cultivar; P07218, P19142, P19143Phaseolus vulgaris (Kidney bean) (French bean); Q7XJC3, Q7XJC4 Pinuspinaster (Maritime pine); Q6UD65 Populus balsamifera subsp.trichocarpa×Populus deltoides; P45731, Q43052, O24266 Populuskitakamiensis (Aspen); Q8H6V5, Q8H6V6 Populus tremuloides (Quakingaspen); P45730 Populus trichocarpa (Western balsam poplar); O64963Prunus avium (Cherry); Q94EN0 Rehmannia glutinosa; P11544 Rhodosporidiumtoruloides (Yeast) (Rhodotorula gracilis); P10248 Rhodotorula rubra(Yeast) (Rhodotorula mucilaginosa); Q9M568, Q9M567 Rubus idaeus(Raspberry); P31425, P31426 Solanum tuberosum (Potato); Q6SPE8 Stellarialongipes (Longstalk starwort); P45732 Stylosanthes humilis (Townsvillestylo); P45734 Trifolium subterraneum (Subterranean clover); Q43210,Q43664 Triticum aestivum (Wheat); Q96V77 Ustilago maydis (Smut fungus);P45735 Vitis vinifera (Grape); and Q8VXG7 Zea mays (Maize).

Numerous studies have focused on the use of the enzyme phenylalanineammonia-lyase (PAL, EC 4.3.1.5) for enzyme substitution treatment ofphenylketonuria (PKU) (Hoskins, et al., Lancet 1(8165):392-394 (1980);Gilbert, et al., Biochem. J. 199(3):715-723 (1981); Hoskins, J. A., etal., Res. Commun. Chem. Pathol. Pharmacol. 35(2):275-282 (1982);Sarkissian, et al., Proc. Natl. Acad. Sci. USA 96(5):2339-2344 (1999);Liu, et al., Artif. Cells Blood Substit. Immobil. Biotechnol.30(4):243-257 (2002); Wieder, et al., J Biol. Chem. 254(24):12579-12587(1979); Gamez, et al., Mol. Ther. 11(6):986-989 (2005); Ambrus, et al.,J. Pharmacol. Exp. Ther. 224(3):598-602 (1983); Ambrus, et al., Science201(4358):837-839 (1978); Kalghatgi, Res. Commun. Chem. Pathol.Pharmacol. 27(3):551-561 (1980); Ambrus, Res. Commun. Chem. Pathol.Pharmacol. 37(1):105-111 (1982); Gilbert, et al., Biochem. Biophys. Res.Commun. 131(2):557-563 (1985); Pedersen, Res. Commun. Chem. Pathol.Pharmacol. 20(3):559-569 (1978); Marconi, et al., Biochimie62(8-9):575-580 (1980); Larue, et al., Dev. Pharmacol. Ther. 9(2):73-81(1986); Ambrus, et al., Ann. Intern. Med. 106(4):531-537 (1987);Bourget, et al., Appl. Biochem. Biotechnol. 10:57-59 (1984); Bourget, etal., FEBS Lett. 180(1):5-8 (1985); Bourget, et al., Biochim. Biophys.Acta 883(3):432-438 (1986); Chang, et al., Artif. Cells Blood Substit.Immobil. Biotechnol. 23(1):1-21 (1995); Chang, et al., Mol. Biotechnol.17(3):249-260 (2001); U.S. Pat. No. 5,753,487).

The use of PAL for cancer treatment has been suggested based on itsability to limit the nutrient supply of phenylalanine to cancer cellsand thereby inhibit neoplastic growth (Fritz, et al., J. Biol. Chem.251(15):4646-4650 (1976); Roberts, et al., Cancer Treat. Rep.60(3):261-263 (1976); Shen, et al., Cancer Res. 37(4):1051-1056 (1977);Shen, et al., Cancer Treat. Rep. 63(6):1063-1068 (1979); Wieder, et al.,J. Biol. Chem. 254(24):12579-12587 (1979)). In addition, PAL-mediatedreduction in phenylalanine prevented the proliferation of murineleukemia and metastatic melanoma. However, intravenously injectedpegylated PAL was cleared rapidly from circulating blood after the 13thinjection (Abell, et al., Cancer Res. 33:2529-2532 (1973); Roberts, etal., (1976), ibid.; Shen, et al., (1977), ibid.; (Shen, et al., J.Reticuloendothelial Soc. 23:167-175 (1978)).

Certain neoplastic or cancer cells have a higher metabolic rate and agreater requirement than normal cells for essential amino acids such asphenylalanine. There is evidence in the literature suggesting thatrestriction or reduction of specific amino acids, e.g., phenylalanine,through the use of amino acid degrading enzymes, e.g., PAL, may reducethe growth of certain tumor cells in human cancer patients and in animalmodels. For example, certain leukemic cells lack the enzyme asparaginesynthetase, which synthesizes the non-essential amino acid asparaginefrom glutamine, and are thus dependent upon asparagine for survival.Oncaspar (pegaspargase, Enzon Pharmaceuticals, Inc.), a pegylatedL-asparaginase, has been used successfully to treat acute lymphoblasticleukemia (ALL) (Graham, Adv. Drug Del. Rev. 55:1293-1302 (2003)). Otherexamples of amino acids as potential targets for enzymatic depletion incancer therapy include glutamine (glutamine deaminase, Medical EnzymesAG), arginine (arginine deiminase, Phoenix Pharmacologics, Inc.) andmethionine (methioninase, Anticancer, Inc.) (See, for example, U.S. Pat.Nos. 6,312,939, 6,737,259 and 5,690,929).

Dietary restriction of phenylalanine has been shown to inhibit growthand metastasis of highly invasive metastatic melanoma and androgenindependent prostate cancer cells in animal models, promote apoptosis oftumor, but not normal, cells in culture, increase survival oftumor-bearing mice, sensitize tumor cells to chemotherapeutic agents,and augment cytotoxicity by toxins (Fu, et al., Nutr. Cancer 31:1-7(1998); Fu, et al., Cancer Res. 59:758-765 (1999); Fu, et al., Nutr.Cancer 45:60-73 (2003); Fu, et al., J. Cell. Physiol. 209:522-534(2006); Meadows, et al., Cancer Res. 42:3056-3063 (1982); Elstad, etal., Anticancer Res. 13:523-528 (1993); Elstad, et al., Nutr. Cancer25:47-60 (1996); Nunez, et al., Cancer Lett. 236:133-141 (2006)).

Enyzmatic depletion of phenylalanine using PAL from the yeastRhodosporidium toruloides (also known as Rhodotorula glutinis) (RtPAL)inhibited the growth of leukemic lymphocytes in culture in vitro (Abell,et al., Cancer Res. 32:285-290 (1972); Stith, et al., Cancer Res.33:966-971 (1973)) and in mice in vivo (Abell, et al., Cancer Res.33:2529-2532 (1973)). However, after repeated injections into mice, theclearance of RtPAL from plasma was greatly accelerated, and theclearance rate was more rapid in tumor bearing, as compared to non-tumorbearing, mice (Fritz, et al., J. Biol. Chem. 251:4646-4650 (1976); Shen,et al., Cancer Res. 37:1051-1056 (1977)). The half-life of RtPAL wasdecreased to about 1 hour after multiple administration due to anincrease in antibody titer, demonstrating that total body radiation maybe necessary to delay clearance and enhance half-life (Shen, et al., J.Reticuloendothelial Soc. 23:167-175 (1978).

RtPAL has been pegylated in an attempt to reduce the enzyme'simmunogenicity and clearance rate in vivo (Wieder, et al., J. Biol.Chem. 254:12579-12587 (1979)). After a single intravenous injection orafter multiple intravenous injections into mice, the blood half-life ofpegylated RtPAL was longer than unpegylated RtPAL; however, thepegylated RtPAL was still rapidly cleared from the blood after thethirteenth intravenous injection.

Although PAL potentially has various therapeutic applications, the useof PAL may be limited by reduced specific activity and proteolyticinstability. Similar to other therapeutic proteins, use of PAL as anenzyme therapy is accompanied by several disadvantages such asimmunogenicity and proteolytic sensitivity (see Vellard, Curr. Opin.Biotechnol. 14:1-7 (2003)). As yet, a concerted effort toward improvingthese parameters has not been made due to a paucity of structural andbiochemical knowledge regarding this protein.

Thus, there remains a need for PAL molecules with optimal kineticcharacteristics, including potent catalytic activity, greater biologicalhalf-life, greater biochemical stability, and/or attenuatedimmunogenicity, for therapeutic use, including the treatment of cancer.

SUMMARY OF INVENTION

The invention is based on the finding that prokaryotic or bacterial PALmay serve as an effective treatment for cancer. The inventioncontemplates compositions of prokaryotic PAL and biologically activefragments, mutants, variants or analogs thereof, with enhancedproperties, such as more potent catalytic activity, greater biochemicalstability and, for therapeutic applications, attenuated immunogenicityand/or greater biological half-life. The invention providespharmaceutical compositions and formulations comprising prokaryotic PALand biologically active fragments, mutants, variants or analogs thereofand a pharmaceutically acceptable carrier, including stabilizers. Thepresent invention also provides methods of production and purificationof prokaryotic PAL and biologically active fragments, mutants, variantsor analogs thereof, and methods of using such compositions fortherapeutic purposes, including the treatment of neoplastic disease andcancer.

As used herein, “bacterial PAL” and “prokaryotic PAL” are usedinterchangeably to mean (1) wild-type PAL from a prokaryotic organism,including but not limited to PAL from Streptomyces maritimus (also knownas EncP, SEQ ID NO:5, FIG. 4), Nostoc punctiforme (SEQ ID NO:2, FIG. 4),Anabaena variabilis (SEQ ID NO:4, FIG. 4), Anacystis nidulans(Lofflehardt, Z. Naturforsch. 31(11-12):693-9 (1976), Photorabdusluminescens TT01 (Williams, et al., Microbiology 151:2543-2550 (2005),and Streptomyces verticillatus (Bezanson, et al., Can. J. Microbiol.16(3):147-51 (1970); (2) fragments, mutants, variants or analogs of suchwild-type PAL enzymes that retain similar (i.e., at least 50%) catalyticactivity for phenylalanine, and that preferably exhibit increasedcatalytic activity, greater biochemical stability, increased half-life,and/or decreased immunogenicity, and (3) chemically modified versions ofsuch wild-type PAL enzymes or fragments, mutants, variants or analogsthereof that have been are linked to other chemical moieties thatprovide other advantageous effects, such as, for example and not forlimitation, enhanced half-life and/or decreased immunogenicity. Forexample, any references to methods of making or using prokaryotic PAL,and fragments, mutants, variants, analogs or chemically modifiedversions thereof, and compositions of such enzyme(s), for therapeuticpurposes, are meant to refer to methods of making, using or formulatingall such wild-type prokaryotic PAL or fragments, mutants, variants,analogs or chemical modifications thereof.

In a first aspect, the present invention provides pharmaceuticalcompositions comprising bacterial PAL and biologically active fragments,mutants, variants or analogs thereof, and a pharmaceutically acceptablecarrier. A preferred embodiment is a bacterial PAL from Nostocpunctiforme (SEQ ID NO:2) or biologically active fragment, mutant,variant or analog thereof. Another preferred embodiment is a bacterialPAL from Anabaena variabilis (SEQ ID NO:4) or biologically activefragment, mutant, variant or analog thereof. The invention contemplatesprokaryotic PAL variants that have greater phenylalanine-convertingactivity and/or reduced immunogenicity as compared to a wild-type PAL.

Preferably, the prokaryotic PAL variants retain the wild-type activesite residues at positions corresponding to Ser210, Ala-Ser-Gly triad(211-213), Asp214, Leu215, Asn270, Val269, Leu266, Leu134, His137,Lys468, Glu496, Gln500 in PAL from Rhodosporidium toruloides PAL (RtPAL)or conservative substitution(s) of these active site residue(s), ofwhich the Ala-Ser-Gly triad at 211-213 is believed to be the bindingsite for phenylalanine.

Desirable prokaryotic PAL variants may include proteins in which one ormore amino acid residues have been substituted by another amino acidresidue to reduce protein aggregation that can be associated withdecreased enzyme activity, increased immunogenicity, and/or otherdisadvantageous effects, such as reduced bioavailability, in vivo. Theinvention contemplates a pharmaceutical composition wherein one or moreamino acid residues of the prokaryotic PAL variant have been substitutedby another amino acid wherein the substitution increasesphenylalanine-converting activity and/or reduces immunogenicity ascompared to the wild-type PAL.

In some embodiments, one or more amino acid residues of the prokaryoticPAL variant have been substituted by another amino acid residue. In someembodiments, one or more cysteine residues of the prokaryotic PALvariant have been substituted by a serine residue. In preferredembodiments, the prokaryotic PAL variant is an Anabaena variabilis PAL(AvPAL). In more preferred embodiments, one or more cysteine residues ofthe AvPAL variant have been substituted by a serine residue selectedfrom the group consisting of cysteine residues at positions 64, 318, 503and 565. In a more preferred embodiment, the cysteine residue atposition 565 of the AvPAL variant has been substituted by a serineresidue. In a most preferred embodiment, the cysteine residues atpositions 503 and 565 of the AvPAL variant have been substituted byserine residues.

Desirable prokaryotic PAL variants may include fusion proteins in whichthe PAL enzyme has been fused to another heterologous polypeptide, suchas a native or modified constant region of an immunoglobulin or afragment thereof that retains the salvage epitope, known in the art toincrease half-life, or is recognized by proteins specific to particularforms of cancer.

The invention further contemplates chemically modified versions of suchprokaryotic PAL polypeptides, which have been linked to a chemicalmoiety that provides other advantageous effects. For example,nonspecific or site-specific (e.g., N-terminal) linkage of water-solublepolymers, e.g., polyethylene glycol, to polypeptides is known in the artto improve half-life, and linkage of chemical moieties may also reduceimmunogenicity and/or improve protease resistance.

In some embodiments, the prokaryotic PAL variant comprises awater-soluble polymer. In preferred embodiments, the prokaryotic PALvariant comprises polyethylene glycol. In a more preferred embodiment,the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) andthe ratio of AvPAL and polyethylene glycol is about 1:3 (1:3 AvPAL:PEG).In a most preferred embodiment, the prokaryotic PAL variant is an AvPALvariant, the ratio of the AvPAL variant and polyethylene glycol is about1:3 (1:3 AvPAL:PEG), and the cysteine residues at positions 503 and 565of the AvPAL variant have been substituted by serine residues.

In some embodiments, one or more amino acid residues of the prokaryoticPAL variant have been substituted by a lysine residue. The pegylation ofan additional lysine residue(s) in a prokaryotic PAL variant can resultin an enzyme that has reduced immunogenicity, increased catalyticactivity, and/or improved biochemical stability. Without being bound toa particular theory, it is hypothesized that a tyrosine residue at/nearthe active site of prokaryotic PAL (e.g., position 78 in AvPAL) can be asite for pegylation, which reduces enzyme activity. In a preferredembodiment, one or more amino acids at/near the active site of theprokaryotic PAL variant that is not required for enzyme activity issubstituted by a lysine residue. Without being bound to a particulartheory, it is hypothesized that pegylation of the substituted lysineresidue at/near the active site sterically hinders a tyrosine residue(e.g., position 78 in AvPAL) from being pegylated.

Such prokaryotic PAL variants are isolated and purified in accordancewith the methods of the present invention and is thereby present inamounts which enable using the prokaryotic PAL enzyme therapeutically.In some embodiments, a cDNA encoding for a complete or wild-typeprokaryotic PAL is used. However, in other embodiments, a cDNA encodingfor a biologically active fragment, mutant, variant or analog thereofmay be used. Further, the present invention provides compositions ofoptimized prokaryotic PAL obtained by structure-based molecularengineering approaches and/or chemically-modified (e.g., pegylated)forms of PAL. Specific embodiments contemplate optimal compositions ofprokaryotic PAL with improved specific activity, enhanced stability,reduced immunogenicity and/or proteolytic sensitivity appropriate fortherapeutic use. A preferred embodiment is a pegylated form of Nostocpunctiforme PAL with improved specific activity, enhanced stability,reduced immunogenicity and/or proteolytic sensitivity. Another preferredembodiment is a pegylated form of Anabaena variabilis PAL with improvedspecific activity, enhanced stability, reduced immunogenicity and/orproteolytic sensitivity.

In some embodiments, the biologically active sites of wild-typeprokaryotic PAL according to the invention may be modified as desired tooptimize PAL kinetic characteristics. In a preferred embodiment, amodified prokaryotic PAL has sufficient activity to reduce plasmaphenylalanine levels in a subject upon treatment to a range from belowthe level of detection to between about 20 μM to 60 μM, preferably toless than about 20 μM, and even more preferably to less than about 10μM, using standard detection methods well known in the art. In otherpreferred embodiments, the biologically active modified prokaryotic PALhas a kcat of at least about 0.1 s-1, preferably greater than about 0.5s-1, and even more preferably greater than about 1.0 s-1. In morepreferred embodiments, the biologically active modified prokaryotic PALhas a kcat of at least about 0.4 s-1, preferably greater than about 2.0s-1, and even more preferably greater than about 4.0 s-1. In otherpreferred embodiments, the biologically active modified prokaryotic PALhas a Km of between about 10 μM to about 2000 μM. In more preferredembodiments, the biologically active modified prokaryotic PAL has a Kmof between about 10 μM to about 1000 μM. In even more preferredembodiments, the biologically active modified prokaryotic PAL has a Kmof between about 10 μM to about 500 μM. In other preferred embodiments,the biologically active modified prokaryotic PAL exhibits enzymaticactivity from about at least 50% of to about 10-fold greater than thewild-type PAL. In other preferred embodiments, the biologically activemodified prokaryotic PAL exhibits enzymatic activity from about at least50% of to about 100% higher than the wild-type PAL. Such biologicalactive modified prokaryotic PAL proteins may be formed using methodswell known in the art, such as by site-directed mutagenesis.

In further embodiments, the invention contemplates use of prokaryoticPAL or a biologically active fragment, mutant, variant or analog thereofthat metabolizes phenylalanine (i.e., converts phenylalanine to anothersubstance) in preparation of a medicament for preventing or treatingcancer in a subject, preferably in a human subject, as well as apharmaceutical composition containing prokaryotic PAL or a biologicallyactive fragment, mutant, variant or analog thereof for use in preventingor treating cancer in a subject, preferably in a human subject. In someembodiments, the medicament is for preventing cancer in a human subject.In other embodiments, the medicament is for treating cancer in a humansubject. In a preferred embodiment, the pharmaceutical compositioncomprises highly purified PAL derived from bacteria, or biologicallyactive fragment, mutant, variant or analog thereof, and apharmaceutically acceptable carrier. Preferred preparations containprokaryotic PAL or biologically active fragment, mutant, variant oranalog thereof with a purity of greater than 90%, 95%, 96%, 97%, 98%,99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. The relative specificactivity of the prokaryotic PAL, or biologically active fragment,mutant, variant or analog thereof, according to the present invention ispreferably at least about 50%, and more preferably greater than about110%, of the specific activity of wild-type prokaryotic PAL.

In a second aspect, the present invention features novel methods ofusing prokaryotic PAL variant compositions for therapeutic purposes. Theinvention contemplates methods of treating various forms of cancer.

In one embodiment, the invention contemplates methods for treatingcancer by administering to a subject in need of such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising a prokaryotic PAL variant and a pharmaceutically acceptablecarrier, wherein the prokaryotic PAL variant has a greaterphenylalanine-converting activity and/or a reduced immunogenicity ascompared to a wild-type PAL and is effective in reducing thephenylalanine concentration in the blood, serum or plasma, preferably inthe plasma, of the subject to a range from below the level of detectionto between about 20 μM to 60 μM, preferably to less than about 20 μM,and even more preferably to less than about 10 μM. In some embodiments,one or more amino acid residues of the prokaryotic PAL variant have beensubstituted by another amino acid residue wherein the substitutionincreases phenylalanine-converting activity and/or reducesimmunogenicity as compared to the wild-type PAL. In some embodiments,one or more cysteine residues of the prokaryotic PAL variant have beensubstituted by another amino acid residue. In some embodiments, one ormore cysteine residues of the prokaryotic PAL variant have beensubstituted by a serine residue. In a preferred embodiment, theprokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant.In a particularly preferred embodiment, one or more cysteine residues ofthe AvPAL variant have been substituted by a serine residue that isselected from the group consisting of cysteine residues at positions 64,318, 503 and 565, by a serine residue at position 565, or by serineresidues at positions 503 and 565. In some embodiments, the prokaryoticPAL variant comprises a water-soluble polymer. In some embodiments, thewater-soluble polymer is polyethylene glycol. In a preferred embodiment,the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL)variant, and the ratio of the AvPAL variant and the polyethylene glycolis about 1:3 (1:3 AvPAL:PEG). In a more preferred embodiment, theprokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant,the ratio of the AvPAL variant and the polyethylene glycol is about 1:3(1:3 AvPAL:PEG), and the cysteine residues at positions 503 and 565 ofthe AvPAL variant have been substituted by serine residues.

In a more particularly preferred embodiment, the invention provides amethod for treating cancer comprising administering to a subject in needof such treatment a therapeutically effective amount of a pharmaceuticalcomposition comprising an AvPAL variant and a pharmaceuticallyacceptable carrier, wherein the cysteine residues at positions 503 and565 of the AvPAL variant have been substituted by serine residues, theAvPAL variant further comprises a water-soluble polymer of polyethyleneglycol, wherein the ratio of AvPAL variant and the polyethylene glycolis about 1:3, and the AvPAL variant is effective in reducing thephenylalanine concentration in the blood, serum or plasma, preferably inthe plasma, of the subject to a range from below the level of detectionto between about 20 μM to 60 μM, preferably to less than about 20 μM,and even more preferably to less than about 10 μM.

In a broad embodiment, the cancer is a cancer wherein the proliferationand/or survival of cells derived from the cancer is sensitive tophenylalanine restriction or depletion. In preferred embodiments, thecancer is lung cancer, brain or central nervous system cancer, coloncancer, prostate cancer, renal cancer or metastatic melanoma. In otherpreferred embodiments, the cancer is head and neck cancer, ovariancancer, uterine cancer, leukemia (e.g., acute myeloid leukemia or acutelymphoblastoid leukemia) or myeloma. In other preferred embodiments, thecancer is pediatric cancer or a resistant cancer (i.e., a cancer thathas been shown to be resistant to cancer therapeutic agents or targetedcancer therapeutic agents).

The invention describes methods of treating cancer in a subjectcomprising administering to the subject a prokaryotic PAL or abiologically active fragment, mutant, variant or analog thereof whereinthe administration of prokaryotic PAL is effective to lower thephenylalanine (Phe) concentration in the blood, serum or plasma,preferably in the plasma, of the subject as compared to theconcentration in the absence of prokaryotic PAL administration. Asubject selected for treatment according to the methods of the inventioncan have any plasma Phe concentration, e.g., from about 40 μM to about2000 μM, or a normal range of plasma Phe concentration, such aconcentration may be in the range from about 40 μM to about 80 μM, moretypically in the range from about 50 μM to about 70 μM, with the rangein most human individuals between about 55 μM to about 65 μM. The plasmaPhe concentration of the subject upon treatment is reduced in the rangefrom below the level of detection to between about 20 μM to 60 μM,preferably to less than about 20 μM, and even more preferably to lessthan about 10 μM, using standard detection methods well known in theart.

The invention also contemplates methods of treating cancer byadministering to a subject in need of such treatment a therapeuticallyeffective amount of a pharmaceutical composition comprising aprokaryotic PAL variant and a pharmaceutically acceptable carrier, incombination with a protein-restricted (i.e., phenylalanine-free) diet,wherein the treatment is effective to produce a decrease in the plasmaPhe concentration of the subject in the absence of the combinedadministration. The plasma Phe concentration of the subject upontreatment is reduced in the range from below the level of detection tobetween about 20 μM to 60 μM, preferably to less than about 20 μM, andeven more preferably to less than about 10 μM, using standard detectionmethods well known in the art.

In another embodiment, prokaryotic PAL or a biologically activefragment, mutant, variant or analog thereof may also be administered incombination with a protein-restricted diet. The protein-restricted dietadministered in the methods herein is one that is aphenylalanine-restricted diet wherein the total Phe intake of thesubject is restricted to less than 600 mg per day. In other embodiments,the protein-restricted diet is a phenylalanine-restricted diet whereinthe total Phe is restricted to less than 300 mg per day. In still otherembodiments, the protein-restricted diet is one supplemented with one ormore amino acids, such as, for example and not for limitation, tyrosine,valine, isoleucine and/or leucine.

Also contemplated is a pharmaceutical composition comprising prokaryoticPAL or a biologically active fragment, mutant, variant or analog thereofand a pharmaceutically acceptable carrier, diluent or excipient. Thepharmaceutical composition may further comprise a medical proteinsupplement. In still other embodiments, the protein supplement isphenylalanine-free. The protein supplement preferably is fortified withL-tyrosine, L-glutamine, L-carnitine at a concentration of 20 mg/100 gsupplement, L-taurine at a concentration of 40 mg/100 g supplement andselenium. It may further comprise the recommended daily doses ofminerals, e.g., calcium, phosphorus and magnesium. The supplementfurther may comprise the recommended daily dose of one or more aminoacids selected from the group consisting of L-leucine, L-proline,L-lysine acetate, L-valine, L-isoleucine, L-arginine, L-alanine,glycine, L-asparagine monohydrate, L-tryptophan, L-serine, L-threonine,L-histidine, L-methionine, L-glutamic acid, and L-aspartic acid. Inaddition, the supplement may be fortified with the recommended dailydosage of vitamins A, D and E. The supplement preferably comprises a fatcontent that provides at least 40% of the energy of the supplement. Sucha supplement may be provided in the form of a powder supplement or inthe form of a protein bar.

The invention also contemplates methods of treating cancer byadministering to a subject in need of such treatment a therapeuticallyeffective amount of a pharmaceutical composition comprising aprokaryotic PAL variant and a pharmaceutically acceptable carrier, incombination with a cancer therapeutic agent or a targeted cancertherapeutic agent, wherein the treatment is effective to produce adecrease in the plasma phenylalanine concentration of the subject in theabsence of the combined administration. The plasma Phe concentration ofthe subject upon treatment is reduced in the range from below the levelof detection to between about 20 μM to 60 μM, preferably to less thanabout 20 μM, and even more preferably to less than about 10 μM, usingstandard detection methods well known in the art.

Preferred embodiments include optimizing the dosage to the needs of theorganism to be treated, preferably mammals or humans, to effectivelyprevent or ameliorate the disease symptoms. Prokaryotic PAL may beadministered in a single daily dose, multiple doses on a daily basis, ina single weekly dose, multiple doses on a weekly basis, in a singlemonthly dose or multiple doses on a monthly basis. In some embodiments,the PAL therapy is not continuous, but rather PAL is administered on adaily basis until the plasma Phe concentration of the subject isdecreased to a range from below the level of detection to between about20 μM to 60 μM, preferably less than about 20 μM, and even morepreferably less than about 10 μM, using standard detection methods wellknown in the art. Preferably, wherein the plasma Phe concentration ofthe subject is monitored on a daily basis and the PAL is administeredwhen a 10%-20% increase in plasma Phe concentration is observed. In yetother preferred embodiments, doses are delivered once weekly. Theinvention contemplates doses of at least 0.001 mg/kg, 0.005 mg/kg, 0.01mg/kg, 0.05 mg/kg, and may range up to 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg,5.0 mg/kg, 12 mg/kg or higher per week. A preferred dose is 1mg/kg/week, a more preferred dose is 0.1 mg/kg/week, and even morepreferred dose is 0.01 mg/kg/week.

A variety of parenteral or nonparenteral routes of administration,including oral, transdermal, transmucosal, intrapulmonary (includingaerosolized), intramuscular, subcutaneous, or intravenous, which deliverequivalent dosages are contemplated. Administration by bolus injectionor infusion directly into the joints or CSF is also specificallycontemplated, such as intrathecal, intracerebral, intraventricular, vialumbar puncture, or via the cisterna magna. Preferably the doses aredelivered subcutaneously or orally.

Other means of increasing prokaryotic PAL activity in the human subjectsare also contemplated, including gene therapy. Transfer of a prokaryoticPAL gene is possible through a variety of means known in the art,including viral vectors, homologous recombination, or direct DNAinjection. Within the scope of this aspect are embodiments featuringnucleic acid sequences encoding all or a part of prokaryotic PAL or abiologically active fragment, mutant, variant or analog thereof, whichmay be administered in vivo into cells that are, for example and not forlimitation, affected with the cancer, located nearby or adjacent to thecancer, hematopoietic cells that circulate in the bloodstream and/ormigrate to the site of the cancer.

In a third aspect, the present invention provides pharmaceuticalcompositions or formulations of prokaryotic PAL variants, comprisingbacterial PAL and biologically active fragments, mutants, variants oranalogs thereof, and a pharmaceutically acceptable carrier, wherein thepharmaceutically acceptable carrier comprises a stabilizer. In someembodiments, the stabilizer is L-phenylalanine or structural analogthereof In some embodiments, the stabilizer is selected from the groupconsisting of L-phenylalanine, trans-cinnamic acid and benzoic acid. Insome embodiments, the stabilizer is L-phenylalanine. In some embodimentsthe stabilizer is trans-cinnamic acid. In some embodiments, thestabilizer is benzoic acid. In a preferred embodiment, the inventionprovides methods of treating cancer using such pharmaceuticalcompositions or formulations.

In a particularly preferred embodiment, the pharmaceutical compositionor formulation comprises a prokaryotic PAL variant and apharmaceutically acceptable carrier, wherein the prokaryotic PAL variantis an AvPAL variant, the ratio of the AvPAL variant and polyethyleneglycol is about 1:3 (1:3 AvPAL:PEG), and the cysteine residues atpositions 503 and 565 of the AvPAL variant have been substituted byserine residues, and the pharmaceutically acceptable carrier comprises astabilizer. In some embodiments, the stabilizer is L-phenylalanine orstructural analog thereof. In some embodiments, the stabilizer isselected from the group consisting of L-phenylalanine, trans-cinnamicacid and benzoic acid. In some embodiments, the stabilizer isL-phenylalanine. In some embodiments the stabilizer is trans-cinnamicacid. In a particularly preferred embodiment, the invention providesmethods of treating cancer using such pharmaceutical compositions orformulations.

In a fourth aspect, the present invention features a method to producerecombinant prokaryotic PAL or a biologically active fragment, mutant,variant or analog thereof in amounts which enable using the enzymetherapeutically. The present invention contemplates PAL derived frombacteria including, but not limited to, Streptomyces, Sorangium,Pseudomonas, and cyanobacteria such as Nostoc and Anabaena. In someembodiments, PAL is derived from the bacterial species Streptomycesmaritimus, S. verticillatus, Soragium cellulosum, Nostoc punctiforme,Nostoc tobacum, Anabaena variabilis, or Pseudomonas putida. In preferredembodiments, PAL is derived from cyanobacteria species Nostocpunctiforme or Anabaena variabilis. In a particularly preferredembodiment, PAL is derived from Anabaena variabilis. In anotherembodiment, prokaryotic PAL enzyme activity is generated using cDNA orDNA sequences that are derived from sequences sometimes described ascoding for HAL activity or featuring a PAL-HAL motif, but possessing keyPAL residues that differ from HAL.

In a broad embodiment, the method comprises the step of transforming acDNA or DNA encoding for all or a part of a prokaryotic PAL or abiologically active fragment, mutant, variant or analog thereof into acell suitable for the expression thereof. In preferred embodiments, anexpression vector is used to transfer the DNA into a suitable cell orcell line for expression thereof. In one particularly preferredembodiment, the cDNA or DNA is transformed into E. coli and recombinantbacterial PAL is overexpressed, optionally as a fusion protein. In afurther embodiment, the method of producing prokaryotic PAL comprisesthe steps of: (a) growing cells transformed with a cDNA or DNA encodingall or a biologically active fragment, mutant, variant or analog thereofof prokaryotic PAL in a suitable growth medium to an appropriate densityto produce a seed culture, (b) introducing the transformed cells into abioreactor, (c) supplying a suitable growth medium to the bioreactor,and (d) separating the transfected cells from the media containing theenzyme.

In a preferred embodiment, recombinant prokaryotic PAL or a biologicallyactive fragment, mutant, variant or analog thereof is over-expressed,with or without an N-terminal tag (e.g., octahistidyl-tag), in a vector,preferably pIBX1 (Su, et al., Appl. Environ. Microbiol. 62:2723-2734(1996)) or pET28a (Invitrogen) with an inducible promoter such as withIPTG (isopropyl-beta-D-thiogalactopyranoside), in E. coli BLR(DE3)/pLysS(Novagen) or E. coli BL21(DE3)/pLysS (Invitrogen) cells. In aparticularly preferred embodiment, the method of producing prokaryoticPAL comprises the steps of: (1) growing a seed culture for abioreactor/fermenter from a glycerol stock in shake flasks; (2)introducing such seed culture into a controlled bioreactor in fed-batchmode; (3) growing said culture in glucose-supplemented media, pH(7.8), >20% dissolved oxygen, agitation up to 1200 rpm, 30° C. untilreaching a cell density of OD600 of 70-100 (˜22-25 hrs); (4) inducingsaid culture with 0.4 mM IPTG; (5) growing said culture at a reducedtemperature of 22 to 26° C. until activity change is <0.1 IU/mL(approximately 40-48 hrs and an OD600 typically of 200); and (5)harvesting bacteria by continuous centrifugation. In a preferredembodiment, the cell culture media is typically defined and composed ofyeast extract protein, peptone-tryptone, glucose, glycerol, casaminoacids, trace salts and phosphate buffering salts.

In a fifth aspect, the present invention features a method to purifyprokaryotic PAL or a biologically active fragment, mutant, variant oranalog thereof. According to a first embodiment, a transformed cell massis grown and ruptured leaving crude recombinant enzyme. Exogenousmaterials are normally separated from the crude bulk to prevent foulingof the columns. Chromatographic purification is conducted using one orseveral chromatographic resins. Subsequently, the purified protein isformulated into a buffer designed to provide stable activity over anextended period of time. In another preferred embodiment, the method topurify the prokaryotic PAL comprises the steps of: (a) lysis of thebacteria containing recombinant PAL; (b) treatment of lysate with heatto inactivate viruses; (c) clarification of this lysate using a secondcontinuous centrifugation step and/or depth filtration; (d) passage ofclarified lysate through a charcoal filtration step; (e) passage offiltrate in (d) through a final filtration step (as with a SartoriousSartopore 0.2 μm filter); (f) passage of final filtrate over ahydrophobic interaction chromatography resin, such as a butylhydrophobic interaction chromatography; (g) passage of eluate in (f)over an anionic chromatography resin, such as a Q ion exchange column;(h) recovery of final product by buffer exchange with tangential flowfiltration; and (i) sterilization of the final product. Those skilled inthe art readily appreciate that one or more of the chromatography stepsmay be omitted or substituted, or that the order of the chromatographysteps may be changed within the scope of the present invention. Finally,appropriate sterilizing steps may be performed as desired.

In a sixth aspect, the present invention contemplates screening assaysfor identifying prokaryotic PAL or a biologically active fragment,mutant, variant or analog thereof that can prevent, ameliorate, or treatcancer by contacting a tumor cell in culture with the prokaryotic PALand determining whether the prokaryotic PAL reduces the proliferationand/or survival of the tumor cells. Such screening assays may alsoinclude the steps of creating variants that include conservative ornon-conservative substitutions in the active sites, e.g. Gly142,Thr-Ser-Gly triad (143-145), Asp146, Leu147, Asn196, Ile195, Leu192,Leu76, Asn79, Met400, Thr428, Gln432 in EncP from Streptomycesmaritimus, or their equivalents in other prokaryotic PAL, such as Nostocpunctiforme or Anabaena variabilis, which are equivalent to residuesSer210, Ala-Ser-Gly triad (211-213), Asp214, Leu215, Asn270, Va1269,Leu266, Leu134, His137, Lys468, Glu496, Gln500 in PAL fromRhodosporidium toruloides (RtPAL), in regions adjacent to the activesites, or throughout the polypeptide sequence, followed by testing thevariants for in vitro phenylalanine converting activity. In certainembodiments, the method is a high throughput assay. In a preferredembodiment, complete genomes of the bacterial species are sequenced andscreened for the presence of prokaryotic PAL homologs using abioinformatics approach. In yet another preferred embodiment, PALcatalytic activity of the protein product of such homologs is confirmed,such as by testing ability to convert phenylalanine to trans-cinnamatein vitro.

In a seventh aspect, the invention provides methods of using prokaryoticPAL compositions for the diagnosis of diseases, including but notlimited to cancer. In one embodiment, prokaryotic PAL is used to measurelevels of Phe in blood, plasma or serum samples. In a furtherembodiment, the invention contemplates a diagnostic kit comprisingprokaryotic PAL for use in monitoring blood, plasma or serum samples ofsubjects for levels of Phe.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1A: Gene sequence of Nostoc punctiforme PAL (SEQ ID NO:1);FIG. 1B: Protein sequence of Nostoc punctiforme PAL (SEQ ID NO:2).

FIG. 2. FIG. 2A: Gene sequence of Anabaena variabilis PAL (SEQ ID NO:3);FIG. 2B: Protein sequence of Anabaena variabilis PAL (SEQ ID NO:4).

FIG. 3. Relatedness tree of aromatic amino acid ammonia-lyases fromprokaryotes and eukaryotes. Sequences were retrieved from GenBank(accession numbers are given in parentheses) and aligned with ClustalX(1.83) using the Neighbor Joining Method.

FIG. 4. Alignment of cyanobacterial protein sequences of N. punctiformePAL (SEQ ID NO:2) and A. variabilis PAL (SEQ ID NO:4) with EncP PAL (SEQID. No. 5) and P. putida HAL (SEQ ID NO:6). Active site residues, whichcorrespond to PAL or HAL activity, are highlighted and underlined.

FIG. 5. FIG. 5A: Protein sequence of Anabaena variabilis phenylalanineammonia-lyase (PAL) with a cysteine to serine substitution at position64 (AvPAL_C64S, SEQ ID NO:7); FIG. 5B: Protein sequence of Anabaenavariabilis PAL with a cysteine to serine substitution at position 318(AvPAL_C318S, SEQ ID NO:8); FIG. 5C: Protein sequence of Anabaenavariabilis PAL with a cysteine to serine substitution at position 503(AvPAL_C503S, SEQ ID NO:9); FIG. 5D: Protein sequence of Anabaenavariabilis PAL with a cysteine to serine substitution at position 565(AvPAL_C565S, SEQ ID NO:10); FIG. 5E: Protein sequence of Anabaenavariabilis PAL with cysteine to serine substitutions at positions 503and 565 (AvPAL_C565SC503S, SEQ ID NO:11). Cysteine to serinesubstitutions are underlined in bold.

FIG. 6. FIG. 6A: Effect of cysteine to serine substitutions at position565 or both positions 565 and 503 of unpegylated AvPAL on in vitro PALspecific enzyme activity after incubation for various lengths of time at37° C. FIG. 6B: Effect of cysteine to serine substitutions at position565 or both positions 565 and 503 of pegylated AvPAL on in vitro PALspecific enzyme activity after incubation for various lengths of time at37° C.

FIG. 7. FIG. 7A: Effect of cysteine to serine substitutions in AvPAL onformation of protein aggregates in solution as analyzed by gelelectrophoresis under denaturing conditions (left panel) or nativeconditions (right panel). FIG. 7B: Effect of cysteine to serinesubstitutions in AvPAL on formation of protein aggregates in solution asanalyzed by SEC-HPLC.

FIG. 8. Effect of cysteine to serine substitutions at positions 565 and503 (dbl Mutant) in AvPAL on site-specific pegylation at various PEGconcentrations.

FIG. 9. Effect of treatment of AvPAL with 0.05% Tween80 or 10 mM EDTA onformation of protein aggregates in solution as analyzed by SEC-HPLC.

FIG. 10. FIG. 10A: Effect of treatment of AvPAL by dithiotreitol (DTT)on formation of protein aggregates in solution as analyzed by SEC-HPLC.FIG. 10B: Effect of treatment of AvPAL by DTT and N-ethylmaleimide (NEM)on formation of protein aggregates in solution as analyzed by SEC-HPLC.

FIG. 11. Effect of phenylalanine (Phe) and trans-cinnamic acid (t-CA) asindicated on the enzyme activity of a pegylated AvPAL with cysteine toserine substitutions at positions 565 and 503 (AvPAL_C565SC503S)(rAV-PAL-PEG) stored for various times (days) at 4° C. (top panel), at25° C. (middle panel) and at 37° C. (bottom panel).

FIG. 12. Effect of tyrosine (Tyr) at 1 and 5 mM as indicated on theenzyme activity of a pegylated AvPAL with cysteine to serinesubstitutions at positions 565 and 503 (AvPAL_C565SC503S) (rAV-PAL-PEG)stored for various times (days) at 4° C. (top panel), at 25° C. (middlepanel) and at 37° C. (bottom panel).

FIG. 13. FIG. 13A: Effect of phenylalanine (Phe), benzoic acid andpyridoxamine, alone or in combination as indicated, on the enzymeactivity of a pegylated AvPAL with cysteine to serine substitutions atpositions 565 and 503 (AvPAL_C565SC503S) (rAV-PAL-PEG) stored forvarious times (weeks) at 4° C. (top panel) and at 37° C. (bottom panel).FIG. 13B: The chemical structures of benzoic acid (left), phenylalanine(middle) and trans-cinnamic acid (right) are depicted.

FIG. 14. FIG. 14A: Effect of a single subcutaneous injection of apegylated AvPAL with cysteine to serine substitutions at positions 565and 503 (AvPAL_C565SC503S) at 4 mg/kg (diamonds) and at 12 mg/kg(squares) into Cynomolgus monkeys on the plasma AvPAL_C565SC503S levelsover time (hours). FIG. 14B: Effect of a single subcutaneous injectionof AvPAL_C565SC503S at 4 mg/kg into Cynomolgus monkeys on the plasmaAvPAL_C565SC503S (diamonds) and phenylalanine (squares) levels over time(hours).

FIG. 15. FIG. 15A: Effect of a single intravenous injection of apegylated AvPAL with cysteine to serine substitutions at positions 565and 503 (AvPAL_C565SC503S) at 1 mg/kg (diamonds), at 5 mg/kg (squares)and at 25 mg/kg (triangles) into rats on the plasma AvPAL_C565SC503Slevels over time (hours). FIG. 15B: Effect of a single subcutaneousinjection of AvPAL_C565SC503S at 10 mg/kg (diamonds), at 25 mg/kg(squares) and at 250 mg/kg (triangles) into rats on the plasmaAvPAL_C565SC503S levels over time (hours).

FIG. 16. FIG. 16A: Effect of a pegylated AvPAL with cysteine to serinesubstitutions at positions 565 and 503 (AvPAL_C565SC503S) at 0.01, 0.1,1, 10 and 100 μg/mL as indicated on proliferation (as measured bypropidium iodide staining) of NOMO1 acute myeloid leukemia (AML) cellsin vitro. FIG. 16B: Effect of AvPAL_C565SC503S at 0.1, 1, 10 and 100μg/mL as indicated on proliferation of IM9 myeloma cells in vitro.

FIG. 17. FIG. 17A: Effect of a pegylated AvPAL with cysteine to serinesubstitutions at positions 565 and 503 (AvPAL_C565SC503S) at 0.01, 0.1,1, 10 and 100 μg/mL as indicated on proliferation (as measured bypropidium iodide staining) of SF268 (top) and 498L (bottom) brain/CNStumor cells in vitro. FIG. 17B: Effect of AvPAL_C565SC503S at 0.01, 0.1,1, 10 and 100 μg/mL as indicated on proliferation of HT29 (top) andHCT116 (bottom) colon tumor cells in vitro. FIG. 17C: Effect ofAvPAL_C565SC503S at 0.01, 0.1, 1, 10 and 100 μg/mL as indicated onproliferation of H460 (top), 529L (middle) and 629L (bottom) lung tumorcells in vitro. FIG. 17D: Effect of AvPAL_C565SC503S at 0.01, 0.1, 1, 10and 100 μg/mL as indicated on proliferation of LNCAP (top), PC3M(middle) and DU145 (bottom) prostate tumor cells in vitro.

DETAILED DESCRIPTION OF THE INVENTION

Several bacterial PAL have been already identified as part of theHAL/PAL family, including but not limited to PAL from Streptomycesmaritimus (also known as EncP, SEQ ID NO:5, FIG. 4), PAL/HAL from Nostocpunctiforme (Accession ZP_(—)00105927 from Nostoc punctiforme ATCC29133, submitted Oct. 1, 2004, NCBI Microbial Genomes AnnotationProject) (SEQ ID NO:2, FIG. 4), PAL/HAL from Anabaena variabilis (GeneID 3679622, Ava_(—)3988 phenylalanine/histidine ammonia-lyase, Anabaenavariabilis ATCC 29413, Mar. 31, 2006) (SEQ ID NO:4, FIG. 4), thephotosynthetic prokaryote Anacystis nidulans (Lofflehardt, Z.Naturforsch. 31(11-12):693-9 (1976)), the gram-negative bacteria fromthe family Enterobacteriaceae, Photorabdus luminescens TT01 (Williams,et al., Microbiology 151:2543-2550 (2005)), and Streptomycesverticillatus (Bezanson, et al., Can. J. Microbiol. 16(3):147-51(1970)). Further, PAL activity has been evaluated in Streptomycesmaritimus (Xiang, et al., J. Biol. Chem. 277:32505-32509 (2002)).Cyanobacteria, such as Anabaena and Nostoc have been studied withrespect to their production of bioactive natural products that aregenerated via mixed polyketide-peptide biosynthetic pathways (Moore,Nat. Prod. Rep. 22(5):580-593 (2005); Becker, et al., Gene 325:35-42(2004); Hoffman, et al., Gene 311:171-180 (2003)).

Although PAL is a ubiquitous higher plant enzyme that catalyzes thenonoxidative deamination of phenylalanine to cinnamic acid in thecommitted step to phenylpropanoid metabolites (Hahlbrock, et al., Annu.Rev. Plant Phys. Plant Mol. Biol. 40:347-369 (1989)), PAL has only beenencountered in a few bacteria where it is involved in benzoyl-CoAbiosynthesis in “S. maritimus” (Xiang, et at, J. Biol. Chem.277:32505-32509 (2002)) and Sorangium cellulosum (Hill, et al., Chem.Commun. 1358-1359 (2003)) and in the biosynthesis of cinnamamide inStreptomyces verticillatus (Bezanson, et al., Can. J. Microbiol.16:147-151 (1970)). The bacteriostatic agent enterocin is a naturalproduct of the marine bacterium “Streptomyces maritimus” whosebiosynthesis involves a number of unusual features (Hertweck, et al.,Chem. Biol. 11:461-468 (2004); Piel, et al., Chem. Biol. 7:943-955(2000); Piel, et al., J. Am. Chem. Soc. 122:5415-5416 (2000); Xiang, etal., Proc. Natl. Acad. Sci. USA 101:15609-15614 (2004)). Among these isthe formation of the rare polyketide synthase (PKS) starter unitbenzoyl-coenzyme A (CoA) (Moore, et al., Nat. Prod. Rep. 19:70-99(2002)). The initial biochemical reaction involves the conversion of theamino acid L-phenylalanine to trans-cinnamic acid by the novel bacterialphenylalanine ammonia-lyase (PAL, EC 4.3.1.5) EncP (Xiang, et al., J.Biol. Chem. 277:32505-32509 (2002)). Activation of cinnamic acid to itsCoA thioester followed by a single round of beta-oxidation yieldsbenzoyl-CoA (Hertweck, et al., Chem. Bio. Chem. 2:784-786 (2001);Hertweck, et al., Tetrahedron 56:9115-9120 (2000); Xiang, et al., J.Bacteriol. 185:399-404 (2003)), which primes the enterocin type II PKSfor chain extension with seven molecules of malonyl-CoA.

The first prokaryotic PAL-encoding gene (encP) (SEQ ID NO:5) wascharacterized and its role in de novo cinnamic acid and enterocinsynthesis in “S. maritimus” was identified (Kalaitzis, et al., J. Am.Chem. Soc. 125:9290-9291 (2003); Xiang, et al., J. Biol. Chem.277:32505-32509 (2002)). The encP gene encodes a 522 amino acid proteinthat is considerably smaller than eukaryotic PALs by nearly 200 aminoacid residues. Although sequence homologous to plant PALs such as fromPetroselinum crispum (Rother, et al., Eur. J. Biochem. 269:3065-3075(2002)) (CAA57056, 30% identical and 48% similar), it rather sharesgreater homology to bacterial histidine ammonia-lyases (HALs, EC4.3.1.3) such as from Pseudomonas putida (Schwede, et al., Biochemistry27:5355-5361 (1999)) (A35251, 36% identical and 54% similar, SEQ IDNO:6, FIG. 4) and to tyrosine ammonia-lyase (TAL) from Rhodobactercapsulatus (Kyndt, et al., FEBS Lett. 512:240-244 (2002)) (FIG. 3). Thehomology includes the conserved active site serine residue at position143 of the phenylalanine/histidine/tyrosine family of ammonia-lyasesthat is the probable precursor of the modified dehydroalanine residue inthe 4-methylideneimidazole-5-one (MIO) prosthetic group (Langer, et al.,Adv. Prot. Chem. 58:175-188 (2001); Poppe, Curr. Opin. Chem. Biol.5:512-524 (2001); Schwede, et al., Biochemistry 27:5355-5361 (1999)).EncP shares greatest sequence homology to AdmH (AAO39102, 63% identicaland 76% similar), a putative phenylalanine aminomutase involved inandrimid biosynthesis in Pantoea agglomerans that is related to thetyrosine aminomutase Sgc4 from Streptomyces globisporus (Christenson, etal., J. Am. Chem. Soc. 125:6062-6063 (2003); Christenson, et al.,Biochemistry 42:12708-12718 (2003)).

HAL and PAL were shown to share in common a mechanism for the chemicallydifficult elimination of ammonia from histidine and phenylalanine,respectively. With both enzymes, a superelectrophilic prosthetic group 5methylene-3,5-dihydroimidazol-4-one (MIO) activates the non-acidic betahydrogen atoms of their respective substrates by a Friedel-Crafts-typeattack at the aromatic ring. The sigma complex that is generatedprevents the extraction of protons from the ring by excluding any basesfrom access to the binding pocket of the enzyme. The formation of anexocyclic double bond is key in the elimination of ammonia,rearomatization, and fragmentation. The prosthetic MIO group isregenerated and the product urocanate or cinnamate is formed (Poppe, etal., Angew. Chem. Int. Ed. 44:3668-3688 (2005)).

Because of the high homology between HAL and PAL, the conserved regionsof HAL and PAL are referred to HAL/PAL conserved region. This highhomology can create some ambiguities in databases like NCBI on thepotential enzyme activity of a “PAL-HAL” protein conducting tomislabeling, such as with protein sequences listed in the NCBI databasefor Nostoc punctiforme and Anabaena variabilis. Therefore some PALenzymes can be mislabeled HAL enzymes. Although the active sites of PALsand HALs are very similar, they are predicted to differ in some keyresidues (Calabrese et al., Biochemistry 43(36):11403-11416 (2004);Xiang et al., (2002) ibid.; Williams et al., (2005) ibid.). Particularlyin HAL, the methionine 383 and glutamic acid 415 from Pseudomonas putida(SEQ ID NO:6) are highly conserved in all HALs but are always replacedin all the PALs described so far (eukaryotic or prokaryotic) by lysineand glutamine respectively (FIG. 4). So it can be said that all proteinswith a “PAL-HAL′” region and having the homologues of lysine 383 andglutamic acid 415 have the sequence signature of a protein with PALactivity. This relatively newly described PAL signature (Williams etal., (2005), ibid.) allows to label properly some enzymes from HAL toPAL and could be used to identify some new PAL enzymes from alreadypublished genes and proteins database.

The present invention relates to compositions of such prokaryotic PALand biologically active fragments, mutants, variants or analogs thereofand their use for therapeutic purposes, including the treatment ofcancer.

A. Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Definition ofstandard chemistry terms may be found in reference works, includingCarey and Sundberg, Advanced Organic Chemistry, 3^(rd) Edition, Vols. Aand B (Plenum Press, New York 1992). The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. See, e.g., T. E. Creighton, Proteins: Structures and MolecularProperties (W.H. Freeman and Company, 1993); A. L. Lehninger,Biochemistry (Worth Publishers, Inc., 4^(th) Edition, 2004); Sambrook,et al., Molecular Cloning: A Laboratory Manual (2^(nd) Edition, 1989);Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press,Inc.); Remington's Pharmaceutical Sciences, 18^(th) Edition (Easton,Pennsylvania: Mack Publishing Company, 1990).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methyiphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is identical to thenucleotide sequence of the polynucleotide-binding partner of the secondpolynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′.

A nucleotide sequence is “substantially complementary” to a referencenucleotide sequence if the sequence complementary to the subjectnucleotide sequence is substantially identical to the referencenucleotide sequence.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide.” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

“Expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,and ligase chain reaction.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).    -   Amino acids may also be grouped as follows:    -   (1) hydrophobic: Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr;    -   (3) acidic: Asp, Glu;    -   (4) basic: Asn, Gln, His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro; and    -   (6) aromatic: Trp, Tyr, Phe.

The terms “identical” or percent “identity,” in the context of two ormore polynucleotide or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm described in prior co-pending U.S. patentapplication Ser. No. 11/230,374 filed on Sep. 19, 2005, which is hereinincorporated by reference in its entirety, or by visual inspection.

The phrase “substantially homologous” or “substantially identical” inthe context of two nucleic acids or polypeptides, generally refers totwo or more sequences or subsequences that have at least 40%, 60%, 80%,90%, 95%, 98% nucleotide or amino acid residue identity, when comparedand aligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection.Preferably, the substantial identity exists over a region of thesequences that is at least about 50 residues in length, more preferablyover a region of at least about 100 residues, and most preferably thesequences are substantially identical over at least about 150 residues.In a most preferred embodiment, the sequences are substantiallyidentical over the entire length of either or both comparisonbiopolymers.

“Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition. In someembodiments, the prokaryotic PAL variant compositions of the inventionare substantially pure or isolated. In some embodiments, the prokaryoticPAL variant compositions of the invention are substantially pure orisolated with respect to the macromolecular starting materials used intheir synthesis. In some embodiments, the pharmaceutical compositions ofthe invention comprise a substantially purified or isolated prokaryoticPAL variant admixed with one or more pharmaceutically acceptableexcipient.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory is naturallyoccurring.

“Wild-type” (wt) is a term referring to the natural genetic form of anorganism. A wild-type is distinguished from a mutant form (an organismwith a genetic mutation).

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein, whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. Such polypeptides may bereferred to as “mutants” herein. These modifications may be deliberate,as through site-directed mutagenesis, or may be accidental, such asthrough mutations arising with hosts that produce the proteins or errorsdue to PCR amplification.

As used herein, “variant,” “analog,” or “derivative” is a compound,e.g., a peptide, having more than about 70% sequence but less than 100%sequence similarity with a given compound, e.g., a peptide. Suchvariants, analogs or derivatives may be comprised of non-naturallyoccurring amino acid residues, including by way of example and notlimitation, homoarginine, ornithine, penicillamine, and norvaline, aswell as naturally occurring amino acid residues. Such variants, analogsor derivatives may also be composed of one or a plurality of D-aminoacid residues, and may contain non-peptide interlinkages between two ormore amino acid residues.

As used herein, the “ratio” of a PAL polypeptide (e.g., AvPAL) and awater-soluble polymer (e.g., polyethylene glycol or PEG) refers to thereaction condition molar ratio between the PAL polypeptide and thewater-soluble polymer. For example, a ratio of about 1:3 for AvPAL andpolyethylene glycol (1:3 AvPAL:PEG) means that the chemically modifiedPAL was produced in a reaction condition with about 1 mol AvPAL per 3mol of polyethylene glycol. Under the reaction conditions described inEXAMPLE 6, infra, a ratio of about 1:3 AvPAL:PEG results in about 10-12mol PEG per mol AvPAL monomer.

“Treatment” or “treating” as used herein refers to prophylactictreatment or therapeutic treatment or diagnostic treatment.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of disease or pathology, i.e., a cancer, orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology. The prokaryotic PAL compositions of the inventionmay be given as a prophylactic treatment to reduce the likelihood ofdeveloping pathology, i.e., a cancer, or to minimize the severity of thepathology, if developed.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology, i.e., a cancer, for the purposeof diminishing or eliminating those signs or symptoms. The signs orsymptoms may be biochemical, cellular, histological, functional,subjective or objective. The prokaryotic PAL compositions of theinvention may be given as a therapeutic treatment or for diagnosis.

“Diagnostic” means identifying the presence or nature of a pathologiccondition, i.e., a cancer. Diagnostic methods differ in theirspecificity and selectivity. While a particular diagnostic method maynot provide a definitive diagnosis of a condition, it suffices if themethod provides a positive indication that aids in diagnosis.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in subject animal, including humans and mammals. Apharmaceutical composition comprises a pharmacologically effectiveamount of a prokaryotic PAL polypeptide and also comprises apharmaceutically acceptable carrier. A pharmaceutical compositionencompasses a composition comprising the active ingredient(s), and theinert ingredient(s) that make up the carrier, as well as any productwhich results, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a prokaryotic PAL polypeptide of thepresent invention and a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical excipients, vehicles, diluents, stabilizers,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers,such as, for example and not for limitation, a phosphate buffered salinesolution, 5% aqueous solution of dextrose, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agentsand/or adjuvants. Suitable pharmaceutical carriers and formulations aredescribed in Remington's Pharmaceutical Sciences, 19th Ed. (MackPublishing Co., Easton, 1995). Preferred pharmaceutical carriers dependupon the intended mode of administration of the active agent. Typicalmodes of administration include enteral (e.g., oral) or parenteral(e.g., subcutaneous, intramuscular, intravenous or intraperitonealinjection; or topical, transdermal, or transmucosal administration). A“pharmaceutically acceptable salt” is a salt that can be formulated intoa prokaryotic PAL variant composition for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of prokaryoticPAL variant of the present invention calculated in an amount sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the novelunit dosage forms of the present invention depend on the particularprokaryotic PAL variant employed and the effect to be achieved, and thepharmacodynamics associated with each prokaryotic PAL variant in thehost.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of themammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish, and the like. The term does not denote a particular ageor gender.

B. Prokaryotic PAL Variants

The elucidation of a reliable three-dimensional structure or structuralmodel for a specific macromolecule permits rational design to become aproductive method for optimization of specific structure and/or functionof said macromolecule. Methods of using a three-dimensional structure orstructural model for optimizing PAL enzymes are described in priorco-pending U.S. patent application Ser. No. 11/230,374 filed on Sep. 19,2005, which is herein incorporated by reference in its entirety. Ahigh-resolution three-dimensional protein crystal structure of aprokaryotic PAL may be used in methods involving protein engineering toimprove the biochemical and biophysical properties of a prokaryotic PAL,and to increase the in vivo therapeutic effectiveness of a prokaryoticPAL. The invention contemplates prokaryotic PAL variants with greaterphenylalanine-converting activity and/or reduced immunogenicity ascompared to a wild-type prokaryotic PAL. The invention also contemplatesprokaryotic PAL variants with greater biochemical stability and/orbiochemical half-life as compared to a wild-type prokaryotic PAL.

Prokaryotic PAL Variants with Enhanced Catalytic Activity

The biologically active sites of a wild-type prokaryotic PAL accordingto the invention may be modified as desired to optimize PAL kineticcharacteristics. Km, the concentration of substrate that giveshalf-maximal activity, is intimately associated with the therapeuticefficacy of PAL in maintaining Phe levels within an acceptable range,i.e., from below the level of detection to between about 20 μM to 60 μM,preferably to less than about 20 μM, and even more preferably to lessthan about 10 μM, using standard detection methods well known in theart. Km is the affinity of the enzyme for the substrate. By controllingaffinity, one can limit or control the efficacy of any enzyme againstsubstrate at different concentrations. For example, if Km is 1000 μM(e.g., PAL from Rhodosporidium toruloides), the activity of the enzymewill be reduced to about 12.5% at blood Phe levels of 240 μM and toabout 3% at blood Phe levels of 60 μM. If Km is 240 μM, the activity ofthe enzyme will be reduced to about 50% at blood Phe levels of 240 μMand to about 12% at blood Phe levels of 60 μM. A preferred therapeuticobjective would be to have a prokaryotic PAL enzyme with sufficientactivity to reduce but also maintain Phe levels upon treatment withinthe range from below the level of detection to between about 20 μM to 60μM, preferably to less than about 20 μM, and even more preferably toless than about 10 μM, using standard detection methods well known inthe art. An enzyme with a Km of about 1000 μM will lose activity rapidlyas Phe levels drop to within normal range (approximately 55-60 μM, seeKaufmann, Proc. Natl. Acad. Sci USA 96:3160-3164 (1999)) and will alsorequire the impractical administration of highly concentrated or largevolumes of doses. An enzyme with a lower Km may rapidly deplete Phe andmaintain Phe levels upon treatment within a range from below the levelof detection to between about 20 μM to 60 μM, preferably to less thanabout 20 μM, and even more preferably to less than about 10 μM, whichmay be useful in the management of cancer.

In preferred embodiments, the biologically active prokaryotic PALvariant has a kcat of at least about 0.1 s-1, preferably greater thanabout 0.5 s-1, and even more preferably greater than about 1.0 s-1. Inmost preferred embodiments, the biologically active prokaryotic PALvariant has a kcat of at least about 0.4 s-1, preferably greater thanabout 2.0 s-1, and even more preferably greater than about 4.0 s-1. Inother preferred embodiments, the biologically active prokaryotic PALvariant has a Km of between about 10 μM to about 2000 μM. In morepreferred embodiments, the biologically active prokaryotic PAL varianthas a Km of between about 10 μM to about 1000 μM. In even more preferredembodiments, the biologically active prokaryotic PAL variant has a Km ofbetween about 10 μM to about 500 μM. In other preferred embodiments, thebiologically active prokaryotic PAL variant exhibits enzymatic activityfrom about 50% of to about 10-fold times greater than the wild-type PAL.In other preferred embodiments, the biologically active prokaryotic PALvariant exhibits enzymatic activity from about 50% of to about 100%higher than the wild-type PAL. Such biological active prokaryotic PALvariants may be formed using methods well known in the art, such as bysite-directed mutagenesis.

Prokaryotic PAL Variants Having Reduced Immunogenicity

A number of strategies are currently used to reduce proteinimmunogenicity. Preferably, modifications that are introduced tominimize the immune response do not destroy the structure, function, orstability of the macromolecule. Effective strategies used includeincreasing human sequence content (chimeras and/or other ‘humanization’approaches), improving solution properties, removing antibody epitopes,introducing chemical derivatization (such as pegylation), and/oridentifying and removing MHC agretopes. For an injected therapeutic, inviva immunoreactivity can be addressed by performing epitope mappingfollowed by rational mutagenesis to modify and/or otherwise mutate thesesites of immunogenicity, alone and in combination with site-specificpegylation (Hershfield, et al., Proc. Natl. Acad. Sci. USA 88:7185-7189(1991); Leong, et al., Cytokine 16(3):106-119 (2001); Lee, et al.,Pharm. Res. 20(5):818-825 (2003)) or other chemical derivatizationmethods to reduce protein immunoreactivity to an acceptable level.Modification of antigenic surface protein regions reduces immunogenicity(Chirino, et al., Drug Discov. Today 9(2):82-90 (2004)). One method ofimprovement involves the construction of smaller sized proteins thatretain catalytic activity (e.g., an absorbance assay is used foractivity measurement). Protein engineering coupled to ELISA screening,can also be used to identify mutants with reduced immunoreactivity.Another method introduces point mutations for additional surface Lyssites for pegylation derivatization, a method shown to reduceimmunogenicity with the test enzyme purine nucleoside phosphorylase(Hershfield, et al. (1991), ibid.). An alternative pathway uses mutationof residues located in protein epitope regions to remove immunogenicsites (Yeung, et al., J. Immunol. 172(11):6658-6665 (2004)). In anapproach that is analogous to antibody humanization, homologous loopregions and/or residues from human antibodies are substituted into thecorresponding loop regions of a homologous protein.

Improving solution properties of proteins may increase specific enzymeactivity and/or reduce immunogenicity. One solution property typical ofbacterially expressed recombinant proteins is the formation of proteinaggregates due, for example, to inter-chain disulfide bind formation,hydrophobic interactions and/or divalent cations (Chi, et al., Pharm.Res. 20(9):1325-1336 (2003)). Aggregation of recombinantly expressedproteins can enhance the immune response (Hermeling, et al., Pharm. Res.21(6):897-903 (2994); Schellekens, Nephrol. Dial. Transplant. 20(suppl6):vi3-9 (2005)). One method of improvement involves substitutingsurface cysteine residues with other amino acid residues (e.g., serine)to minimize the possibility of formation of inter-chain disulfide bonds.For example, substitution of two surface cysteine residues with serineresidues reduced the aggregation of chorismate lyase with minor effectson enzyme activity (Holden, et al., Biochim. Biophys. Acta1594(1):160-167 (2002)). The invention contemplates prokaryotic PALvariants having one or more cysteine residues substituted by anotheramino acid residue, preferably a serine residue. In some embodiments,one or more cysteine residues of the prokaryotic PAL are substituted byanother amino acid residue. In preferred embodiments, the prokaryoticPAL is AvPAL. In more preferred embodiments, one or more cysteineresidues of AvPAL are substituted by a cysteine residue.

C. Chemically Modified Prokaryotic PAL Variants

Macromolecule chemical modification can be performed in a non-specificfashion (leading to mixtures of derivatized species) or in asite-specific fashion (based on wild-type macromoleculereactivity-directed derivatization and/or site-selective modificationusing a combination of site-directed mutagenesis and chemicalmodification) or, alternatively, using expressed protein ligationmethods (Hofmann, et al., Curr. Opin. Biotechnol. 13(4):297-303 (2002)).Preferably, chemical modification is used to reduce immunogenicity.Pegylation is a demonstrated method to reduce immunogenicity of proteins(Bhadra, et al., Pharmazie 57(1):5-29 (2002)), but glycosylation andother chemical derivatization procedures, using modification withphosphorylation, amidation, carboxylation, acetylation, methylation,creation of acid-addition salts, amides, esters, and N-acyl derivativesare also possible (Davis, Science 303:480-482 (2004)). Methods forpegylating PAL proteins and for determining the optimal degree ofpegylation are described in prior co-pending U.S. patent applicationSer. No. 11/230,374 filed on Sep. 19, 2005, which is herein incorporatedby reference in its entirety. The invention contemplates prokaryotic PALvariants comprising a water-soluble polymer (i.e., polyethylene glycolor PEG).

The invention contemplates introducing one or more lysine residuesat/near the active site of a prokaryotic PAL variant to enhancecatalytic activity, reduce immunogenicity and/or improve biochemicalstability, in part by blocking potential pegylation of other amino acidresidues (e.g., tyrosine) at/near the active site of the enzyme or byblocking potential pegylation of a lysine residue important for enzymeactivity. Without being bound to a particular theory, it is hypothesizedthat a tyrosine residue at/near the active site of a prokaryotic PAL(i.e., position 78 or 314 in AvPAL) can be a site for pegylation, whichreduces enzyme activity. In some embodiments, one or more amino acidresidues at/near the active site of the prokaryotic PAL, which are notrequired for enzyme activity, are substituted by a lysine residue. In apreferred embodiment, the prokaryotic PAL is AvPAL. In a more preferredembodiment, the AvPAL tyrosine residue at position 78 or 314 is notaccessible for pegylation. Again without being bound to a particulartheory, it is hypothesized that a lysine residue of a prokaryotic PAL(i.e., position 419 in AvPAL), which is normally blocked from pegylationdue to pegylation of a neighboring lysine residue PAL (i.e., position413 in AvPAL), can be a site for pegylation, which reduces substratebinding and/or catalytic activity. In some embodiments, one or moreamino acid residues of the prokaryotic PAL are substituted by a lysineresidue, such that a lysine residue important for the enzyme's substratebinding and/or catalytic activity is not accessible for pegylation. In apreferred embodiment, the prokaryotic PAL is AvPAL. In a more preferredembodiment, the AvPAL lysine residue at position 419 is not accessiblefor pegylation.

Pegylated Prokaryotic PAL Variants

Examples 7 through 9 of prior co-pending U.S. patent application Ser.No. 11/451,999 filed on Jun. 12, 2006, which is herein incorporated byreference in its entirety, describe the effects of pegylated andnonpegylated forms of lysine mutant R91K PAL from Rhodosporidiumtoruloides (RtPAL), PAL produced by the cyanobacterium Nostocpunctiforme (NpPAL), and PAL produced by the cyanobacterium Anabaenavariabilis (AvPAL) on phenylalanine (Phe) levels in the ENU2 orBTBR^(enu2) mouse. This animal model is a homozygous mutant at thephenylalanine hydroxylase gene (PAH) locus resulting in an animal withsevere hyperphenylalanemia. The high plasma Phe levels make this animalthe appropriate model for evaluating the ability of PAL to reduce plasmaPhe. Administration of pegylated forms of NpPAL and AvPAL resulted ingreater reduction in Phe in the ENU2 mice as compared to unpegylatedNpPAL and AvPAL, respectively. Such effects were maintained for NpPALupon weekly injections over a ten-week period. These results show thatpegylation of PAL from the cyanobacteria, Nostoc punctiforme andAnabaena variabilis, is essential in reducing Phe levels in PKU affectedmice.

Example 14 of prior co-pending U.S. patent application Ser. No.11/451,999 filed on Jun. 12, 2006, which is herein incorporated byreference in its entirety, describe the effect of serine substitution ofthe cysteine residues (e.g., at positions 503 and 565) in the AvPALpolypeptide on Phe levels in ENU2 mice. The administration of thepegylated AvPAL double cysteine mutant AvPAL_C565SC503S resulted in areduction in plasma Phe that was comparable to that achieved withpegylated wild-type AvPAL. In addition, the anti-PAL antibody titerswere lower in animals injected with pegylated AvPAL variant as comparedto pegylated wild-type AvPAL. These results show that a pegylated AvPALvariant has (1) in vivo PAL enzyme activity that is comparable to thepegylated wild-type AvPAL, and (2) has reduced immunogenicity comparedto the pegylated wild-type AvPAL.

D. Therapeutic Uses and Administration of Prokaryotic PAL Variants 1.Various Forms of Cancer

The present invention is directed to the treatment of cancer withmethods that comprise the use of prokaryotic PAL compositions, eitheralone or in combination with other therapeutic regimens, for example andnot for limitation, cancer therapeutic agents or targeted cancertherapeutic agents. In particular, it is contemplated that prokaryoticPAL compositions may be used to treat that patient population withblood, serum or plasma, phenylalanine (Phe) concentrations at any level(e.g., from about 40 μM to and 2000 μM), where the normal range in humanplasma is between about 55 μM and 60 μM (Kaufman, Proc. Natl. Acad. Sci.USA 96:3160-3164 (1999)).

A “cancer therapeutic agent” as used herein refers to any compound,e.g., small molecule or peptide/polypeptide, which has been shown toexert a therapeutic effect (i.e., inhibition of proliferation and/orsurvival) on cancer cells. Typically, the cancer therapeutic agent is acytotoxic agent or a cytostatic agent.

A “targeted cancer therapeutic agent” as used herein refers to anycompound, e.g., small molecule or peptide/polypeptide, or polypeptide orconjugated polypeptide that has been shown to exert a therapeutic effect(i.e., inhibition of proliferation and/or survival) on specific cancercells or tissues. Typically, the targeted cancer therapeutic agent is anantibody, a polypeptide having an antibody-like domain, or otherpolypeptide, e.g., enzyme, hormone, growth factor, cytokine, etc., whichselectively binds to the surface of a target cell. The antibody,polypeptide having an antibody-like domain, or other polypeptide may beunconjugated or may be conjugated to a cancer therapeutic agent. Thetargeted cancer therapeutic agent can be a compound that exerts atherapeutic effect on specific cancer cells or tissues.

The prokaryotic PAL compositions of the present invention are useful fortreating any cancer for which Phe restriction or depletion inhibits itsproliferation and/or survival. The identification of cancers for whichtreatment with the prokaryotic PAL compositions of the present inventionmay be useful can be made on the basis of in vitro culture experiments(see EXAMPLE 14) or in vivo human tumor xenograft studies in mice (seeEXAMPLE 15) using, for example, a tumor biopsy specimen, or bycomparison with tumor types with known or demonstrated sensitivity toPhe restriction or depletion in animal models of human cancer. Inpreferred embodiments, the cancer is lung cancer, brain or centralnervous system cancer, colon cancer, prostate cancer, renal cancer ormetastatic melanoma. In other preferred embodiments, the cancer is headand neck cancer, uterine cancer, leukemia (e.g., acute myeloid leukemiaor acute lymphoblastic leukemia) or myeloma. In other preferredembodiments, the cancer is pediatric cancer or a resistant cancer (i.e.,a cancer that has been shown to be resistant to cancer therapeuticagents or targeted cancer therapeutic agents).

Certain embodiments of the present invention are directed to treatingcancer by administering to the subject a composition comprisingprokaryotic PAL or a biologically active fragment, mutant, variant oranalog thereof in combination with a protein-restricted (i.e.,phenylalanine-free) diet, wherein the combined administration of theprokaryotic PAL and the protein-restricted diet is effective to lowerthe Phe concentration in the blood, plasma or serum of said subject ascompared to the concentration in the absence of said combinedadministration.

It is contemplated that the methods of the invention will entailmonitoring the plasma Phe concentration of the individual to be treatedby prokaryotic PAL compositions. The patient is then treated byadministering prokaryotic PAL compositions alone, or in combination withother therapeutic regimens, such as cancer therapeutic agents ortargeted cancer therapeutic agents, or a combined regimen of prokaryoticPAL and a protein-restricted (i.e., phenylalanine-free) diet, such thatthere is produced at least a 10% decrease in the blood, plasma or serumPhe concentrations of the patient. Preferably, the method will produceat least a 25%, and more preferably at least a 50% decrease in theblood, plasma or serum Phe concentration. Even more preferably, themethod will produce at least a 50%, 60%, 70%, 80%, 90%, 95% or greaterdecrease in the blood, serum or plasma Phe concentration of theindividual (for example, where a patient with a plasma Phe concentrationof 60 μM is treated, a 50%, 70% or 90% decrease in the Phe concentrationwill produce a plasma Phe concentration of 30 μM, 18 μM or 6 μM,respectively). Of course, it should be understood that the treatmentmethods of the present invention should attempt to lower the blood,serum or plasma Phe concentrations of the patient upon treatment to arange from below the level of detection to between about 20 μM to 60 μM,preferably to less than about 20 μM, and even more preferably to lessthan about 10 μM, using standard detection methods well known in theart.

Parenteral, oral, or other standard routes of administration and dosagecan be achieved using standard methods.

2. Compositions for Use in the Treatment

The present invention contemplates therapeutic intervention of cancer.Such intervention is based initially on the use of prokaryotic PALcompositions, pharmaceutical compositions and formulations, which may beused alone or in combination with other therapeutic regimens, such ascancer therapeutic agents or targeted cancer therapeutic agents, or acombined regimen of a low protein diet (i.e., low phenylalanine) andprokaryotic PAL, or both. Further, prokaryotic PAL and/or dietaryrestrictions may be further combined with other therapeutic compositionsthat are designed, for example, to combat manifestations of lowphenylalanine levels, such as, for example and not for limitation,tyrosine supplementation. This section provides a discussion of thecompositions, pharmaceutical compositions and formulations that may beused in the treatments contemplated herein.

Prokaryotic PAL Compositions, Pharmaceutical Compositions andFormulations

In general, the present invention contemplates pharmaceuticalcompositions comprising therapeutically effective amounts of prokaryoticPAL compositions of the invention together with one or morepharmaceutically acceptable excipients, vehicles diluents, stabilizers,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such pharmaceutical compositions include diluents of various buffercontent (e.g., Tris-HCl, phosphate), pH and ionic strength; additivessuch as detergents and solubilizing agents (e.g., Polysorbate 20,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); see, e.g., Remington'sPharmaceutical Sciences, 18^(th) Edition (1990, Mack Publishing Co.,Easton, Pa.) pages 1435:1712, which are herein incorporated byreference. An effective amount of active ingredient is atherapeutically, prophylactically, or diagnostically effective amount,which can be readily determined by a person skilled in the art by takinginto consideration such factors as body weight, age, and therapeuticgoal.

The prokaryotic PAL pharmaceutical compositions of the present inventionmay include a buffering agent to maintain the pH of the solution withina desired range. Preferred buffering agents include Tris-HCl, sodiumacetate, sodium phosphate, and sodium citrate. Mixtures of thesebuffering agents may also be used. The amount of buffering agent usefulin the composition depends largely on the particular buffer used and thepH of the solution. For example, acetate is a more efficient buffer atpH 5 than pH 6 so less acetate may be used in a solution at pH 5 than atpH 6. A more preferred buffering agent is Tris-HCl. A preferred pH rangefor the pharmaceutical compositions of the present invention is about pH6.0-8.5. A more preferred pH range for the pharmaceutical compositionsof the present invention is about pH 7.0-8.0. A most preferred pH rangefor the pharmaceutical compositions of the present invention is about pH7.0-7.6.

The pharmaceutical compositions of the present invention may furtherinclude an isotonicity-adjusting agent to render the solution isotonicand more compatible for injection. A preferred agent is sodium chloridewithin a concentration range of 50-200 mM. A more preferred agent issodium chloride within a concentration range of 100-150 mM. A mostpreferred agent is sodium chloride within a concentration range of130-150 mM.

Pharmaceutically acceptable carriers or excipients may includestabilizers, which are molecules that stabilize the prokaryotic PALcompositions of the invention. The term “stabilize” as used herein, ismeant to include, for example and not for limitation, increasing theshelf-life of a prokaryotic PAL enzyme, protecting the prokaryotic PALenzyme from proteolytic digestion, maintaining the prokaryotic PALenzyme in an active conformation, and preserving the prokaryotic PALenzyme activity upon storage at elevated temperatures.

Stabilizers of the present invention include L-phenylalanine (Phe) andstructural analogs thereof, such as trans-cinnamic acid (t-CA), benzoicacid, tyrosine (Tyr), and the like. Loss of activity of a plant PAL fromPhaseolus vulgaris (PvPAL) has been shown upon removal of its substrateL-phenylalanine after affinity purification (Da Cunha, Eur. J. Biochem.178:243-248 (1988)), and a yeast PAL from Rhodosporidium toruloides(RtPAL) has been shown to be protected from protease inactivation bytyrosine (Wang, et al., Mol. Genet. Metab. 86:134-140 (2005); Pilbak, etal., FEBS J. 273:1004-1019 (2006)). As shown herein below, Phe andcertain of its structural analogs have the ability to stabilize PEG:PALconjugates of a prokaryotic PAL from Anabaena variabilis (AvPAL) (seeEXAMPLE 11). Without being bound to a particular theory, it ishypothesized that the prokaryotic PAL enzyme is more stable as anenzyme-substrate complex, wherein the bound substrate Phe is convertedto the product t-CA or to a transition state analog of t-CA. The t-CAremains bound to the otherwise highly reactive active site center (MIOgroup), thereby stabilizing the PAL enzyme. Accordingly, the PAL enzymesubstrate, Phe, product, t-CA, or structural analogs thereof arestabilizers of the invention.

The invention contemplates a pharmaceutical composition comprising aprokaryotic PAL variant and a pharmaceutically acceptable carrier,wherein the pharmaceutically acceptable carrier comprises a stabilizer.The stabilizer is Phe or structural analog thereof. The stabilizer isselected from the group consisting of L-phenylalanine, trans-cinnamicacid and benzoic acid. A preferred range for the stabilizers of theinvention is from about 0.1 to 20 moles of stabilizer per mole activesite of prokaryotic PAL. A more preferred range for the stabilizers ofthe invention is from about 0.5 to 10 moles of stabilizer per moleactive site of prokaryotic PAL. A most preferred range for thestabilizers of the invention is from about 1 to 10 moles of stabilizerper mole active site of prokaryotic PAL.

In some embodiments, the pharmaceutical composition comprises aprokaryotic PAL variant and a pharmaceutically acceptable carrier,wherein the prokaryotic PAL variant has a greaterphenylalanine-converting activity and/or a reduced immunogenicity ascompared to a wild-type PAL and is effective in reducing the Pheconcentration in the blood, serum or plasma of the subject to a rangefrom below the level of detection to between about 20 μM to 60 μM,preferably to less than about 20 μM, and even more preferably to lessthan about 10 μM, and wherein the pharmaceutically acceptable carriercomprises a stabilizer. In some embodiments, the stabilizer is Phe orstructural analog thereof. In some embodiments, the stabilizer isselected from the group consisting of L-phenylalanine, trans-cinnamicacid and benzoic acid.

In preferred embodiments, the pharmaceutical composition comprises aprokaryotic PAL variant and a pharmaceutically acceptable carrier,wherein the prokaryotic PAL variant is an Anabaena variabilis PAL(AvPAL) variant, wherein the cysteine residues at positions 503 and 565of the AvPAL variant have been substituted by serine residues, the AvPALvariant further comprises a water-soluble polymer of polyethyleneglycol, wherein the ratio of AvPAL variant and the polyethylene glycolis about 1:3; and the AvPAL variant is effective in reducing thephenylalanine concentration in the blood, serum or plasma of the subjectto a range from below the level of detection to between about 20 μM to60 μM, preferably to less than about 20 μM, and even more preferably toless than about 10 μM, and wherein the pharmaceutically acceptablecarrier comprises a stabilizer. In some embodiments, the stabilizer isPhe or structural analog thereof. In preferred embodiments, thestabilizer is selected from the group consisting of L-phenylalanine,trans-cinnamic acid and benzoic acid.

As used herein, when contemplating prokaryotic PAL variant compositions,the term “therapeutically effective amount” refers to an amount that iseffective to produce the intended beneficial effect on health of acancer patient. In some embodiments, a therapeutically effective amountof a prokaryotic PAL variant gives a decrease in blood, plasma or serum,preferably plasma, L-phenylalanine levels that provides benefit to thepatient. The amount will vary from one individual to another and willdepend upon a number of factors, including the overall physicalcondition of the patient, diet and disease state. The amount ofprokaryotic PAL used for therapy gives an acceptable decrease in blood,plasma or serum, preferably plasma, L-phenylalanine levels, andmaintains this value during PAL treatment at a beneficial level(typically in a range from less than about 5% to between about 35% and100%, preferably in a range from less than about 5% to about 35%, andeven more preferably in a range from less than about 5% to about 15% ofthe normal range of blood, plasma or serum, preferably plasma,L-phenylalanine). In some embodiments, a therapeutically effectiveamount of a prokaryotic PAL variant reduces tumor growth, tumor size ortumor burden by greater than about 10%, 30%, 50%, 70%, 90%, 95%, 98% or99% in a treated patient as compared to an untreated patient. In someembodiments, a therapeutically effective amount of a prokaryotic PALvariant maintains the tumor in static condition in a treated patient ascompared to an untreated patient. In some embodiments, a therapeuticallyeffective amount of a prokaryotic PAL variant increases survival time ordisease-free time at least about 10%, 20%, 50%, 100%, 2-fold, 5-fold or10-fold longer in a treated patient as compared to an untreated patient.A therapeutically effective amount of the prokaryotic PAL variantcompositions of the invention may be readily ascertained by one skilledin the art using publicly available materials and procedures.

The invention provides for administering prokaryotic PAL variants lessfrequently than native PAL. The dosing frequency will vary dependingupon the condition being treated, but in general will be about one timeper week. It is understood that the dosing frequencies actually used mayvary somewhat from the frequencies disclosed herein due to variations inresponses by different individuals to the prokaryotic PAL variants; theterm “about” is intended to reflect such variations. It is contemplatedthat the prokaryotic PAL variants are administered about two times perweek, about one time per week, about one time every two weeks, about onetime per month, or longer than about one time per month.

The present invention may thus be used to reduce blood, plasma or serumL-phenylalanine levels. Numerous cancer-related conditions, wheredepletion of blood, plasma or serum L-phenylalanine levels would bebeneficial, may be treated with the prokaryotic PAL variantpharmaceutical compositions of the invention.

The prokaryotic PAL pharmaceutical compositions prepared in accordancewith the present invention are preferably administered by parenteralinjection, either intravenously, intraperitoneally, subcutaneously,intramuscularly, intraarterially or intrathecally. However, it would beclear to one skilled in the art that other routes of delivery could alsobe effectively utilized using the pharmaceutical compositions of thepresent invention.

The methods described herein use prokaryotic PAL pharmaceuticalcompositions comprising the molecules described above, together with oneor more pharmaceutically acceptable excipients, vehicles, diluents,stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers, and optionally other therapeutic and/or prophylacticingredients. Such excipients include liquids such as water, saline,glycerol, polyethylene glycol, hyaluronic acid, ethanol, cyclodextrins,modified cyclodextrins (i.e., sufobutyl ether cyclodextrins), etc.Suitable excipients for non-liquid formulations are also known to thoseof skill in the art.

Pharmaceutically acceptable salts can be used in the compositions of thepresent invention and include, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. A thorough discussion of pharmaceuticallyacceptable excipients and salts is available in Remington'sPharmaceutical Sciences, 18^(th) Edition (Easton, Pa.: Mack PublishingCompany, 1990).

Additionally, auxiliary substances, such as wetting or emulsifyingagents, biological buffering substances, surfactants, and the like, maybe present in such vehicles. A biological buffer can be virtually anysolution which is pharmacologically acceptable and which provides theformulation with the desired pH, i.e., a pH in the physiologicallyacceptable range. Examples of buffer solutions include saline, phosphatebuffered saline, Tris buffered saline, Hank's buffered saline, and thelike.

Depending on the intended mode of administration, the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, creams, ointments, lotions or the like,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the prokaryotic PAL in combination with apharmaceutically acceptable carrier and, in addition, may include otherpharmaceutical agents, adjuvants, diluents, buffers, etc.

In general, the prokaryotic PAL pharmaceutical compositions of thisinvention will be administered as pharmaceutical formulations includingthose suitable for oral (including buccal and sub-lingual), rectal,nasal, topical, pulmonary, vaginal or parenteral (includingintramuscular, intraarterial, intrathecal, subcutaneous and intravenous)administration or in a form suitable for administration by inhalation orinsufflation. The preferred manner of administration is intravenoususing a convenient daily dosage regimen, which can be adjusted accordingto the degree of affliction.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc., a prokaryotic PAL variant composition as described herein andoptional pharmaceutical adjuvants in an excipient, such as, for example,water, saline, aqueous dextrose, glycerol, ethanol, and the like, tothereby form a solution or suspension. If desired, the pharmaceuticalcomposition to be administered may also contain minor amounts ofnontoxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, tonicifying agents, and the like, for example, sodiumacetate, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate. etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, referenced above.

For oral administration, the composition will generally take the form ofa tablet, capsule, or softgel capsule, or may be an aqueous ornonaqueous solution, suspension or syrup. Tablets and capsules arepreferred oral administration forms. Tablets and capsules for oral usewill generally include one or more commonly used carriers such aslactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. When liquid suspensions are used, the activeagent may be combined with emulsifying and suspending agents. Ifdesired, flavoring, coloring and/or sweetening agents may be added aswell. Other optional components for incorporation into an oralformulation herein include, but are not limited to, preservatives,suspending agents, thickening agents, and the like.

Parenteral formulations can be prepared in conventional forms, either asliquid solutions or suspensions, solid or lyophilized forms suitable forreconstitution, solubilization or suspension in liquid prior toinjection, or as emulsions. Preferably, sterile injectable suspensionsare formulated according to techniques known in the art using suitablecarriers, dispersing or wetting agents and suspending agents. Thesterile injectable formulation may also be a sterile injectable solutionor a suspension in a nontoxic parenterally acceptable diluent orsolvent. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils, fatty esters or polyols areconventionally employed as solvents or suspending media. In addition,parenteral administration may involve the use of a slow release orsustained release system such that a constant level of dosage ismaintained.

The prokaryotic PAL compositions of the invention described herein canbe administered to a patient at therapeutically effective doses to treatcancer. The toxicity and therapeutic efficacy of such prokaryotic PALcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, such as, for example, bydetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀ /ED₅₀. Prokaryotic PALcompositions exhibiting large therapeutic indices are normallypreferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosagepreferably lies within a range of circulating concentrations thatinclude the ED₅₀ with little or minimal toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration utilized. The therapeutically effective dose or amountcan be determined from cell culture assays, and from animal models.

Dietary Protein

In addition to administering prokaryotic PAL compositions to cancerpatients, it is contemplated that the dietary protein of the patientsalso may be restricted or modified. Those of skill in the art are awareof various commercially available protein formulas for use in thetreatment of PKU. Such formulas include MAXIMAID, PHENEX 1, PHENEX 2(Ross Laboratories, Liverpool, UK), LOFENALAC, PHENYL-FREE(Mead-Johnson), and the like.

Those of skill in the art may use the referenced protein formulas, whichare generally free of Phe concentrations. The protein formulas often aresupplemented with amino acids that are deficient in PKU patients. Suchamino acids include, for example, L-tyrosine, and L-glutamine.

Further, as it is known that L-carnitine and taurine, which are normallyfound in human milk and other foodstuffs of animal origin, also shouldbe supplied in addition to the protein restriction. In certainembodiments, the L-carnitine may be supplied as 20 mg/100 g of proteinsupplement, and the taurine may be supplied as 40 mg/100 g proteinsupplement in order to help supply amounts of these factors normallyfound in human milk and foods of animal origin.

In addition, those of skill in the art are referred to the 2000 NationalAcademy of Sciences-National Research Council Dietary Reference Intakesfor a further listing of other components, such as essential vitaminsand minerals that should be supplied to the patient to ensure that othersupplements are being provided despite the dietary protein restriction.

Referring to the discussion above regarding total protein amounts anddesirable plasma Phe concentrations, one of skill in the art will beable to determine the amount of dietary protein restriction that isrequired and thus adjust the diet of the patient accordingly. Uponadministering prokaryotic PAL to that subject, determining whether themethods of the invention are effective will entail determining theplasma Phe concentrations of the patient on a regular basis to ensurethat the plasma Phe concentrations remain in a range from below thelevel of detection to between about 20 μM to 60 μ, preferably to lessthan about 20 μM, and even more preferably to less than about 10 μM.Tests for determining such concentrations are described below.Preferably, concentrations of less than the level of detection tobetween about 20 μM to 60 μM are achieved, more preferably to less thanabout 20 μM, and even more preferably to less than about 10 μM.

In certain embodiments, the invention provides a method for treatingcancer comprising administering to a subject in need of such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising a prokaryotic phenylalanine ammonia-lyase (PAL) variant and apharmaceutically acceptable carrier, wherein the PAL variant has agreater phenylalanine-converting activity and/or a reducedimmunogenicity as compared to a wild-type PAL, and is effective inreducing the phenylalanine concentration in the blood, serum or plasmaof the subject to a range from below the level of detection to betweenabout 20 μM to 60 μM, preferably to less than about 20 μM, and even morepreferably to less than about 10 μM, and further comprisingadministering to the subject a protein-restricted (i.e.,phenylalanine-free) diet.

Certain methods of the invention involve the combined use of prokaryoticPAL and dietary protein restriction to effect a therapeutic outcome inpatients with various forms of cancer. To achieve the appropriatetherapeutic outcome in the combination therapies contemplated herein,preferably one would generally administer to the subject the prokaryoticPAL composition and the dietary restriction in a combined amounteffective to produce the desired therapeutic outcome (i.e., a loweringof plasma Phe concentration to a range from below the level of detectionto optimally about 20 μM to 60 μM, preferably to less than about 20 μM,and even more preferably to less than about 10 μM., using standarddetection methods well known in the art). This process may involveadministering the prokaryotic PAL composition and the dietary proteintherapeutic composition at the same time. This may be achieved byadministering a single composition or pharmacological proteinformulation that includes all of the dietary protein requirements andalso includes the prokaryotic PAL within said protein formulation.Alternatively, the dietary protein (supplement or normal protein meal)is taken at about the same time as a pharmacological formulation(tablet, injection or drink) of prokaryotic PAL. Prokaryotic PAL alsomay be formulated into a protein bar or other foodstuff such asbrownies, pancakes, cake, suitable for ingestion.

As the administration of prokaryotic PAL would not generate tyrosine(unlike administration of PAH), such treatment will still result intyrosine being an essential amino acid for such patients. Therefore,dietary supplementation with tyrosine may be desirable for patientsreceiving prokaryotic PAL alone in combination with the dietary proteintherapy.

In other alternatives, prokaryotic PAL treatment may precede or followthe dietary protein therapy by intervals ranging from minutes to hours.In embodiments where the protein and the prokaryotic PAL compositionsare administered separately, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that PAL will still be able to exert an advantageouslyeffect on the patient. In such instances, it is contemplated that onewould administer the PAL within about 2-6 hours (before or after) of thedietary protein intake, with a delay time of only about 1 hour beingmost preferred. In certain embodiments, it is contemplated that PALtherapy will be a continuous therapy where a daily dose of PAL isadministered to the patient indefinitely.

3. Combination Therapy

Further, in addition to therapies based solely on the delivery ofprokaryotic PAL and dietary protein regulation, the methods of thepresent invention also contemplate combination therapy with acomposition that specifically targets one or more of the symptoms ofcancer. Such compositions include, for example and not for limitation,cancer therapeutic agents and targeted cancer therapeutic agents.

Cancer Therapeutic Agents

In accordance with the methods described herein, any agent that exerts atherapeutic effect on cancer cells (i.e., inhibition of proliferationand/or survival) can be used as the cancer therapeutic agent incombination with the prokaryotic PAL compositions of the invention.Typically, the cancer therapeutic agent is a cytotoxic agent or acytostatic agent.

The cancer therapeutic agent can be administered as a cancer co-therapywith the prokaryotic PAL compositions of the invention. “Cancerco-therapy” as used herein means that the cancer therapeutic agent andthe prokaryotic PAL composition are administered simultaneously orsequentially, either the cancer therapeutic agent followed by theprokaryotic PAL composition, or vice versa.

Useful classes of cytotoxic agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and-carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, and the like.

Individual cytotoxic agents include, for example, an androgen,anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin,busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine(BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

In some embodiments, the cancer therapeutic agent is a cytotoxic agent.Suitable cytotoxic agents include, for example, dolastatins (e.g.,auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g.,enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel anddocetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In certain embodiments, the cytotoxic agent is a conventionalchemotherapeutic such as, for example, doxorubicin, paclitaxel,melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. Inaddition, potent agents such as CC-1065 analogues, calicheamicin,maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can beused.

In certain embodiments, the cytotoxic agent is a DNA minor groovebinding agent, for example, a CBI compound or an enediyne (e.g.,calicheamicin).

In certain embodiments, the cancer therapeutic agent is an anti-tubulinagent. Examples of anti-tubulin agents include, but are not limited to,taxanes (e.g., TAXOL (paclitaxel), TAXOTERE (docetaxel)), T67 (Tularik),vinca alkyloids (e.g., vincristine, vinblastine, vindesine, andvinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB,AEVB). Other antitubulin agents include, for example, baccatinderivatives, taxane analogs (e.g., epothilone A and B), nocodazole,colchicine and colcimid, estramustine, cryptophysins, cemadotin,maytansinoids, combretastatins, discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents, for example, maytansine or DM-1.

In certain embodiments, the cancer therapeutic agent is a radioisotope.In certain other embodiments, the cancer therapeutic agent is not aradioisotope.

In certain embodiments, the cytotoxic agent is an antimetabolite. Theantimetabolite can be, for example, a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

In other embodiments, the cytotoxic agent is tacrolimus, cyclosporine orrapamycin. In further embodiments, the cytoxic agent is aldesleukin,alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine,anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone,capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukindiftitox, dexrazoxane, dromostanolone propionate, epirubicin, Epoetinalfa, estramustine, exemestane, Filgrastim, floxuridine, fludarabine,fulvestrant, gemcitabine, gemtuzumab ozogamicin, goserelin, idarubicin,ifosfamide, imatinib mesylate, Interferon alfa-2a, irinotecan,letrozole, leucovorin, levamisole, meclorethamine or nitrogen mustard,megestrol, mesna, methotrexate, methoxsalen, mitomycin C, mitotane,nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate,pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman,plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase,Rituximab, Sargramostim, streptozocin, tamoxifen, temozolomide,teniposide, testolactone, thioguanine, toremifene, Tositumomab,Trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine,vincristine, vinorelbine and zoledronate.

In certain embodiments, the invention provides a method for treatingcancer comprising administering to a subject in need of such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising a prokaryotic phenylalanine ammonia-lyase (PAL) variant and apharmaceutically acceptable carrier, wherein the PAL variant has agreater phenylalanine-converting activity and/or a reducedimmunogenicity as compared to a wild-type PAL, and is effective inreducing the Phe concentration in the blood, serum or plasma of thesubject to a range from below the level of detection to between about 20μM to 60 μM, preferably to less than about 20 μM, and even morepreferably to less than about 10 μM, and further comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a cancer therapeutic agent.

Targeted Cancer Therapeutic Agents

In accordance with the methods described herein, any agent that exerts atherapeutic effect on specific cancer cells (i.e., inhibition ofproliferation and/or survival) can be used as the targeted cancertherapeutic agent in combination with the prokaryotic PAL compositionsof the invention. Typically, the targeted cancer therapeutic agent is anantibody or an enzyme or protein that binds selectively to particulartype of tumor cells or tissues, for example and not for limitation, byvirtue of having an antibody-like targeting domain or viareceptor-mediated binding. The antibody, polypeptide having anantibody-like targeting domain, enzyme or protein can be radiolabeled,or can be conjugated to a toxin or other agent that is able to exert acytotoxic or cytostatic effect on the targeted cells or tissues.Alternatively, the targeted cancer therapeutic agent can be an agentthat exerts a therapeutic effect on specific cancer cells or tissues(i.e., inhibition of proliferation and/or survival) by virtue ofinhibiting or activating a protein, for example and not for limitation,an enzyme or a receptor, which is preferentially expressed or active inthat particular type of tumor cell or tissue.

The targeted cancer therapeutic agent can be administered as a targetedcancer co-therapy with the prokaryotic PAL compositions of theinvention. “Targeted cancer co-therapy” means that the targeted cancertherapeutic agent and the prokaryotic PAL composition are administeredsimultaneously or sequentially, either the targeted cancer therapeuticagent followed by the prokaryotic PAL composition, or vice versa.

In certain embodiments, the targeted cancer therapeutic agent is ahumanized anti HER2 monoclonal antibody, RITUXAN (rituximab; Genentech;a chimeric anti CD20 monoclonal antibody); OVAREX (AltaRex Corporation,MA); PANOREX (Glaxo Wellcome, NC; a murine IgG2a antibody); CetuximabErbitux (Imclone Systems Inc., NY; an anti-EGFR IgG chimeric antibody);Vitaxin (MedImmune, Inc., MD; Campath I/H (Leukosite, MA; a humanizedIgG1 antibody); Smart MI95 (Protein Design Labs, Inc., CA; a humanizedanti-CD33 IgG antibody); LymphoCide (Immunomedics, Inc., NJ; a humanizedanti-CD22 IgG antibody); Smart ID10 (Protein Design Labs, Inc., CA; ahumanized anti-HLA-DR antibody); Oncolym (Techniclone, Inc., CA; aradiolabeled murine anti-HLA-Dr10 antibody); Allomune (BioTransplant,CA; a humanized anti-CD2 mAb); Avastin (Genentech, Inc., CA; ananti-VEGF humanized antibody); Epratuzamab (Immunomedics, Inc., NJ andAmgen, CA; an anti-CD22 antibody); and CEAcide (Immunomedics, NJ; ahumanized anti-CEA antibody).

Other suitable antibodies include, but are not limited to, antibodiesagainst the following antigens: CA125, CA 15-3, CA19-9, L6, Lewis Y,Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase,prostate specific antigen, prostatic acid phosphatase, epidermal growthfactor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, anti transferrin receptor, p97,MUC1-KLH, CEA, gp100, MART1, Prostate Specific Antigen, IL-2 receptor,CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin,P21, MPG, and Neu oncogene product.

In certain embodiments, the targeted cancer therapeutic agent is anenzyme or other protein having an antibody-like targeting domain orhaving the ability to selectively bind to particular cells or tissues(e.g., by ligand-receptor binding). Typically, protein having anantibody-like targeting domain, enzyme or other protein is conjugated toa cancer therapeutic agent, for example, a compound orpeptide/polypeptide, such as a toxin e.g., ricin, Diptheria toxin,Pseudomonas endotoxin, and the like (Kreitman, AAPS J. 8(3):E532-E551(2006)) or other agent that is able to exert a cytotoxic or cytostaticeffect on the targeted cells or tissues.

In certain embodiments, the targeted cancer therapeutic agent iscompound (e.g., small molecule or peptide/polypeptide) that exerts atherapeutic effect on specific cancer cells or tissues, e.g., aserine/threonine kinase inhibitor, a tyrosine kinase inhibitor, anuclear receptor agonist, a nuclear receptor antagonist, and the like.

In certain embodiments, the invention provides a method for treatingcancer comprising administering to a subject in need of such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising a prokaryotic phenylalanine ammonia-lyase (PAL) variant and apharmaceutically acceptable carrier, wherein the PAL variant has agreater phenylalanine-converting activity and/or a reducedimmunogenicity as compared to a wild-type PAL, and is effective inreducing the Phe concentration in the blood, serum or plasma of thesubject to a range from below the level of detection to between about 20μM to 60 μM, preferably to less than about 20 μM, and even morepreferably to less than about 10 μM, and further comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a targeted cancer therapeutic agent.

4. Identifying and Monitoring Patient Populations

As discussed herein throughout, it will be necessary in variousembodiments of the present invention to determine whether a given cancerpatient may be responsive to prokaryotic PAL therapy, and to determinethe phenylalanine (Phe) concentrations of the patient both initially andduring an ongoing therapeutic regimen to monitor the efficacy of theregimen in terms of reduction in plasma Phe concentration. Exemplarysuch methods are described herein below.

Identifying Patients Responsive to Prokaryotic PAL Therapy

The identification of patients for which treatment with the prokaryoticPAL compositions of the present invention may be useful can be made onthe basis of in vitro culture experiments or in vivo human tumorxenograft studies in mice, or by comparison with tumor types with knownor demonstrated dependence on Phe for its growth and its sensitivity toPhe restriction or depletion in animal models of human cancer.

In vitro culture experiments, in which tumor cells (e.g., from a tumorbiopsy or clinical aspirate) are grown in the absence or presence of aprokaryotic PAL composition or in the absence or presence of aphenylalanine-deficient medium, can be performed using protocols knownin the art (see, for example, Abell, et al., Cancer Res. 32:285-290(1972); Stith, et al., Cancer Res. 33:966-971 (1973); Fu, et al., CancerRes. 59:758-765 (1999); Fu, et al., J. Cell. Physiol. 209:522-534(2006); Elstad, et al., Nutr. Cancer 25:47-60 (1996); Nunez, et al.,Cancer Lett. 236:133-141 (2006)). See also EXAMPLE 14.

In vivo human tumor xenograft studies in mice, in which tumor cells(e.g., from a tumor biopsy or clinical aspirate) are injected into nudeor SCID mice, and then transplanted into naïve nude or SCID mice in theabsence or presence of a prokaryotic PAL composition or in the absenceor presence of a phenylalanine-deficient diet, can be performed usingprotocols known in the art (see, for example, Abell, et al., Cancer Res.33:2529-2532 (1973); Shen, et al., Cancer Res. 37:1051-105 (1977); Fu,et al., Nutr. Cancer 31:1-7 (1998); Fu, et al., Nutr. Cancer 45:60-73(2003); Meadows, et al., Cancer Res. 42:3056-3063, 1982; Elstad, et al.,Anticancer Res. 13:523-528, (1993)). See also EXAMPLE 15.

Tumor types with known or demonstrated dependence on Phe for its growthand its sensitivity to Phe restriction or depletion in animal models ofhuman cancer include, for example, leukemia, metastatic myeloma, andandrogen-independent prostate cancer (Abell, et al., (1973), ibid.;Shen, et al., (1977), ibid.; Fu, et al., (1998), ibid.; Fu, et al.,(2003), ibid.; Meadows, et al., (1982), ibid.; Elstad, et al., (1993),ibid.).

EXAMPLE 14 shows that the proliferation and/or survival of tumor cellsderived from lung cancer, brain or central nervous system cancer, coloncancer, prostate cancer, renal cancer, metastatic melanoma, head andneck cancer, uterine cancer, leukemia (e.g., acute myeloid leukemia oracute lymphoblastoid leukemia) and myeloma is sensitive to Pherestriction upon incubation with a prokaryotic PAL composition of thepresent invention.

Determination of Phenylalanine Concentrations

Methods for determining the concentration of phenylalanine (Phe) inblood, serum or plasma are known in the art, some of which are describedin prior co-pending U.S. patent application Ser. No. 11/230,374 filed onSep. 19, 2005, which is herein incorporated by reference in itsentirety. It is contemplated that the plasma Phe levels of the patientswill be monitored at convenient intervals (e.g., daily, every other dayor weekly) throughout the time course of the therapeutic regimen. Bymonitoring the plasma Phe levels with such regularity, the clinicianwill be able to assess the efficacy of the treatment and adjust theprokaryotic PAL and/or dietary protein requirements accordingly.

E. Production of Prokaryotic Pal

Another aspect of the invention is a method of producing prokaryotic PALor biologically active fragment, mutant variant or analog thereof. In apreferred embodiment, recombinant prokaryotic PAL or a biologicallyactive fragment, mutant, variant or analog thereof is over-expressed,with or without an N-terminal tag (e.g., octahistidyl-tag), in a vector,preferably pIBX1 (Su, et al., Appl. Environ. Microbiol. 62:2723-2734(1996)) or pET28a (Invitrogen) with an inducible promoter such as withIPTG (isopropyl-beta-D-thiogalactopyranoside), in E. coli BLR(DE3)/pLysS(Novagen) or E. coli BL21(DE3)/pLysS (Invitrogen) cells. Seed culturefor a bioreactor/fermenter is grown from a glycerol stock in shakeflasks. Such seed culture is then used to spike into a controlledbioreactor in fed-batch mode. Glucose is supplemented and pH iscontrolled with base (NH4OH) and agitation is up to 1200 rpm. O₂ feedkeeps dissolved oxygen to greater than 20%. The cells are grown at atemperature of 37° C. until reaching an OD₆₀₀ of 70-100 (-22-25 hrs) andthen induced with 0.4 mM IPTG. The temperature is reduced to 30° C. andgrown until activity change is <0.1 IU/mL (approximately 40-48 hrs andan OD₆₀₀ typically of 200). Cell culture media is typically defined andcomposed of yeast extract protein, peptone-tryptone, glucose, glycerol,casamino acids, trace salts and phosphate buffering salts. Therecombinant prokaryotic PAL product or biologically active fragment,mutant, variant or analog thereof is produced intra-cellularly and notsecreted. The bacteria are harvested by continuous centrifugation(Alfa-Laval, Carr, Ceba, or equivalent).

F. Purification of Prokaryotic Pal

A further aspect of the present invention features a method to purifyprokaryotic PAL or a biologically active fragment, mutant, variant oranalog thereof. According to a first embodiment, a transformed cell massis grown and ruptured leaving crude recombinant enzyme. Exogenousmaterials are normally separated from the crude bulk to prevent foulingof the columns. Chromatographic purification is conducted using one orseveral chromatographic resins. Subsequently, the purified protein isformulated into a buffer designed to provide stable activity over anextended period of time. In another preferred embodiment, the method topurify the prokaryotic PAL or biologically active fragment, mutant,variant or analog thereof comprises: (a) lysis of the bacteriacontaining recombinant prokaryotic PAL or biologically active fragment,mutant, variant or analog thereof using a pressure homogenizer (butpotentially by other physical means such as glass bead lysis); (b) heattreatment; (c) clarification of this lysate using a second continuouscentrifugation step and/or depth filtration (as with Cuono Zeta Plus orMaximizer, Pall Filtron, or Millipore Millistak or Opticao filters); (d)passage through a charcoal filtration step (as with Millipore Millistak40AC); (e) passage through a final filtration step (as with a SartoriousSartopore 0.2 μm filter); (f) passage over a butyl hydrophobicinteraction chromatography (as in Toyopearl Butyl 650M from TosohBiosciences); (g) passage over a Q ion exchange column (as in aMacroprep High Q from BioRad); and (h) recovery of final product bybuffer exchange with tangential flow filtration (as with a SartoriousHydrosart or PES 100 kDa membrane). Those skilled in the art readilyappreciate that one or more of the chromatography steps may be omittedor substituted, or that the order of the chromatography steps may bechanged within the scope of the present invention. Finally, appropriatesterilizing steps may be performed as desired.

Having now generally described the invention, the same may be morereadily understood through the following reference to the followingexamples. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Examples Example 1 Cloning of Nostoc Punctiforme and Anabaena VariabilisPAL DNA Manipulations

N. punctiforme genomic DNA was purchased from ATCC (29133D) and the PALgene (ZP_(—)00105927) was PCR-amplified from primers5′-CACTGTCATATGAATATAACATCTCTACAACAGAACAT-3′ (SEQ ID NO:12) and5′-GACAGTGGCGGCCGCTCACGTTGACTTTAAGCTCGAAAAAATATG-3′ (SEQ ID NO:13). Theresulting PCR product was digested with NdeI and NotI and the 1.7 kbfragment was ligated into pET-28a(+) and pET-30a(+) (Novagen) for N-Histagged and untagged, respectively.

A. variabilis cells were purchased from ATCC (29413). Genomic DNA wasextracted (Qiagen) and the PAL gene (YP_(—)324488) was amplified bySOE-PCR to remove an NheI site. Primer 1(5′-CACTGTGCTAGCATGAAGACACTATCTCAAGCACAAAG-3′) (SEQ ID NO:14) and primer2 (5′-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3′) (SEQ IDNO:15) were used to amplify nucleotides 1-1190 and primer 3(5′-CCACTAATTGATGTTGATAACCAAGCCAGCTATCATGGAGGAAATTTCC-3′) (SEQ ID NO:16)and primer 4 (5′-CACTGTGCGGCCGCTTAATGCAAGCAGGGTAAGATATCTTG-3′) (SEQ IDNO:17) were used to amplify nucleotides 1142-1771. These two PCRproducts were combined to amplify the full-length gene with primers 1and 4. The resulting PCR product was digested with NheI, blunted withKlenow (NEB), then digested with NotI. The 1.7 kb fragment was ligatedinto pET-28a(+) and pET-30a(+) (Novagen). This plasmid was named3p86-23.

The A. variabilis PAL (AvPAL) gene was also cloned into the vector pIBX7(Tkalec, et al., Appl. Environ. Microbiol. 66:29-35 (2000)), which wasderived from pIBX1 (Su, et al., Appl. Environ. Microbiol. 62:2723-2734(1996)) (see EXAMPLE 7).

Bacterial Strains and Culture Conditions

For N. punctiforme PAL (NpPAL), E. coli BL21(DE3) cells (Stratagene)were transformed with pGro7 (TaKaRa) and competent BL21(DE3)pGro7 cellswere prepared by the Inoue Method (Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3^(rd) Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, 2001)). These cells weretransformed with pET-28-NpPAL and cultured in 25 mL LB with 50 mg/Lkanamycin and 20 mg/L chloramphenicol overnight at 37° C. Twentymilliliters of this culture was seeded into 1 L of LB medium withkanamycin, chloramphenicol, and 500 mg/L L-arabinose and grown at 37° C.At an OD₆₀₀ of 0.6, the culture was chilled on ice. After 5 minutes, theculture was induced with 0.3 mM IPTG and grown for 16 hours at 20° C.Cells were harvested by centrifugation.

BL21(DE3)pLysS cells (Stratagene) were transformed with AvPAL andcultured identically to NpPAL without the arabinose induction.

AvPAL cloned in the pIBX7 vector (see EXAMPLE 7) was introduced bytransformation into BLR(DE3)/pLysS (Novagen) cells and cultured in 25 mLLB with 50 mg/L kanamycin overnight at 37° C. Twenty milliliters of thisculture was seeded into 1 L of LB medium with kanamycin, and grown at37° C. At an OD₆₀₀ of 0.6, the culture was chilled on ice. After 5minutes, the culture was induced with 0.3 mM IPTG and grown for 16 hoursat 30° C. Cells were harvested by centrifugation.

Example 2 Purification of NpPAL and AvPAL

The cultures were centrifuged in a bench-top centrifuge at 5,000 g for20 minutes and the supernatant discarded. The cell pellets weretypically frozen at −70° C. prior to further processing. Upon thawing,the cell pellets were suspended to approximately 80 optical densityunits (600 nm) in TBS (25 mM Tris, 150 mM NaCl, pH 7.8). The cells werelysed by two passes through an APV pressure homogenizer at 12-14,000psi. The crude lysate was then heat-treated at 55° C. for 2 hours. Thelysate is centrifuged at 10,000 g for 30 minutes and the supernatantretained and filtered with a 0.2 μm vacuum filter (Corning).

The PAL was purified from the clarified lysate by passage sequentiallyover a butyl 650M column (Tosoh BioSciences) and a MacroPrep High Qcolumn (BioRad). The eluted product showed a high level of purity byboth SDS PAGE and reverse phase HPLC.

Example 3 Generation of Pegylated PAL Variants

A method for pegylation of PAL from Rhodosporidium toruloides (RtPAL) isdescribed below. Similar methods are used for pegylation of prokaryoticPAL (e.g., Nostoc punctiforme (NpPAL) or Anabaena variabilis (AvPAL))are described in EXAMPLE 6.

Protein Pegylation

Pegylation uses modifications of literature methods (Hershfield, et al.,(1991), ibid.; U.S. Pat. No. 6,057,292; Lu, et al., Biochemistry40(44):13288-13301 (2001); Nektar Therapeutics, 2003 catalog). ActivatedPEGs include both the linear PEG succinimidyl succinates (mPEG-SPA, MW 5kDa or MW 20 kDa) and the branched PEG hydrosuccinimides (mPEG₂-NHSester, MW 10 kDa or MW 40 kDa), which are both capped on one end with amethoxy group and available from Nektar Therapeutics; experimentaldetermination of optimal pegylated proteins is normally required(Veronese, et al., J. Bioactive Compatible Polymers 12:196-207 (1997)).Optimal pegylation conditions are determined using different ratios ofPAL:PEG (taking into account the molar ratio of protein along with thenumber of lysines per protein monomer), different pHs, differentbuffers, various temperatures and incubation times. High PAL protein:PEGderivatization ratios are necessary since native PAL has a large numberof lysines (29 per Rhodosporidium toruloides (Rt) monomer) and becauseun-modified PAL displays immunoreactivity upon repeated injection inmice and since naked (wild-type) PAL is quickly inactivated uponexposure to proteases. Pegylation reactions are stopped by freezing at−20° C., and the samples will be analyzed by SDS-PAGE, MALDI-TOF massspectroscopy, activity assessment, proteolytic sensitivity, andimmunoreactivity.

Prior to activity, proteolysis, and immune assessment, and in order toremove excess unreacted PEG, reactions are dialyzed against pH 8.5, 0.05M potassium phosphate buffer overnight at 4° C. with stirring usingTube-O-Dialyzers (GenoTechnology). After protein concentration isdetermined using the NI protein assay kit (GenoTechnology), PAL activitymeasurements will be performed on underivatized and PEG derivatized PALsamples using standard reaction conditions, as previously described.Following in vitro characterization, in vivo trials will be conductedwith the most promising pegylated therapeutic candidates using the PKUmouse model.

Characterization

Protein concentration is determined using the PAL extinction coefficient(0.5 and 0.75 mg mL⁻¹ cm⁻¹ for RtPAL and AvPAL, respectively) at 280 nmfor non-modified protein samples and for pegylated protein samples theconcentration is calculated using the NI Protein Assay (GenoTechnology)that includes sample processing to remove non-protein contaminants thatmight interfere with accurate protein concentration determination.

PEG-PAL products are characterized by peptide mapping techniques todetermine site-specific pegylation (LC/ESI-MSD), and trinitrobenzenesulfonate (TNBS) to determine the free amine titration before and afterpegylation. Peptide mapping determines the relative occupancy ofpegylation at a majority of the tryptic peptides that terminate withlysine, however, due to size and multiple adjacent lysine trypticpeptides, not all sites are visible using this technique. The TNBS assaymore accurately defines the average number of PEG molecules per mol ofenzyme, but gives no information about which sites get pegylated. Forthis reason, both assays are used and are complementary to each other.Rough estimates of percent derivatization of PAL products by PEG can bedetermined by SDS-PAGE and native gel analyses. Enzymatic assays areused to assess specific activity before and after pegylation and toprovide evidence that there is no loss of the tetrameric PAL structure.

PAL Activity Assay

The PAL activity assay is conducted using a Cary UV spectrophotometer(Cary 50) in the kinetics mode. The activity of PAL with L-phenylalaninesubstrate is assayed at room temperature (25° C.) by measuring theproduction of trans-cinnamate monitored by the absorbance increase at290 nm (Hodgins, (1968), ibid.). The molar extinction coefficient oftrans-cinnamic acid at 290 nm is 10,238 liter cm⁻¹. Reaction mixturescontain 22.5 mM phenylalanine in 100 mM Tris-HCl buffer, pH 8.5. Forstandard measurements the final enzyme concentration is 0.0035 mg/mL,but for kinetic studies the enzyme concentration in the assay isadjusted so that the slope at 290 nm per min is in the range of 0.005 to0.02. Activity data is expressed as specific activity (μmol×min⁻¹ mg⁻¹).One unit of PAL is defined as that amount of enzyme that produces 1 μmolof trans-cinnamic acid per minute at room temperature.

Example 4 Test of In Vitro Half-Life and Immunogenicity

After biochemical characterization, the most promising PEG-PALcandidates are screened for immunoreactivity against antibodies raisedby PKU mice injected with native PAL (non-pegylated) using threedifferent and complementary techniques (Western blot, ELISA, andimmunoprecipitation (IP)).

For Western blot analysis, PAL anti-serum (from mice injected withnative PAL) is used in a dilution 1:10,000. As a negative control theserum from buffer treated-mice is also used in the same dilution. Thesecondary antibody, alkaline phosphatase-conjugated goat anti-mouse IgG(Promega), is diluted to 1:5,000 and color is developed using the APsubstrate Western Blue (Promega). The ELISA test is performed usingNunc/Immuno Maxisorp plates (Nalge Nunc International) followingstandard procedures using 1 mg/mL of PAL in PBS and blocking with PBS,0.05% Tween-20, 2% BSA. The mouse antisera (from native PAL exposedmice) is diluted 1:10,000 in EB block solution (PBS, 0.05% Tween-20, 2%BSA), and a HRP-goat anti-mouse IgG is used as secondary antibody withTMB used for detection at 450 nm.

Immunoprecipitation is used to test for PAL antibody binding. Proteinsamples (PAL or pegylated PAL) are incubated in TTBS buffer (Trisbuffered saline with 0.1% Tween) and PAL activity is measured beforeadding the antibody sample. Each sample is incubated with 8-fold excessof positive control anti-PAL serum and a duplicate negative controlreaction using non-immune mouse serum. After incubation, protein GSepharose 4 (50%, v/v) is added in excess, taking into account the mouseIgG binding capacity of the beads, and the samples are incubated againat 4° C. overnight with rotation. Supernatants are recovered bycentrifugation and the PAL activity of each sample is assayed on thesupernatants. The bead pellets are not discarded, so that furtheranalysis by Western blot can be performed. To confirm that antibody-beadbinding has occurred, Western blot is used to detect the PAL antigen onthe beads. Beads that have been recovered by centrifugation after thePAL binding step are washed several times with TTBS and TBS buffers.Following these rinses, SDS-PAGE loading buffer is added to the beadsand the samples are heated at 95° C. for 5 minutes. Samples are thenanalyzed by Western blot using PAL anti-serum. Enzyme variants showingpoor antibody binding have corresponding little PAL in the pelleted beadfractions as detected by Western blot and show higher activitiesremaining in the supernatant as compared to native un-modified PAL whichdisplays high antibody binding.

Example 5 Test of Protease Sensitivity

Protease mapping studies on native PAL from R. toruloides have indicatedprimary sites of proteolytic sensitivity. Removal of such sites mayreduce or eliminate proteolytic sensitivity and contribute to thedevelopment of an effective PKU enzyme substitute. However, eliminationof such sites for proteolytic sensitivity may result in the reduction orloss of enzyme activity.

After protein engineering has created improved PAL (and PEG-PAL) mutantsthat retain activity, screening for protease resistance using incubationwith a trypsin/chymotrypsin protease cocktail, followed by monitoringfor retention of activity (via OD₂₉₀ measurement) and reduced proteincleavage (via PAGE gel analysis) allows for the identification ofmutants with appropriate in vitro properties to be used for in vivotesting.

Proteolytic stability will be assessed using incubation with a proteasecocktail that approximates the intestinal environment and contains 2.3mM trypsin, 3.5 mM chymotrypsin, 3.05 mM carboxypeptidase A, and 3.65 mMcarboxypeptidase B. Proteolysis testing will involve enzymaticincubations, adding proteases to the PAL solutions, to determine thedegree of protease sensitivity for the different protein variants beingexamined (native or mutant protein with or without pegylation or otherchemical modification), including time courses of activity retention andstability retention after protease exposure. SDS-PAGE and MALDI-TOF massspectrometric mapping experiments will be used to determine the locationof any protease sensitive sites (Kriwacki, R. W., et al., J. Biomol.Tech. 9(3):5-15 (1980)). These mapping results will be important todetermine primary sites of protease susceptibility (such as the twoprimary sites already identified), so that all major sensitivity sitescan be removed using pegylation protection and/or mutation to removeand/or protect susceptible regions from the PAL architecture.

Example 6 Generation of PEGylated NpPAL and AvPAL

In general, PEGylation for both NpPAL and AvPAL involves mixing theprotein with SUNBRIGHT ME-200HS 20 kDa NHS-activated PEG (NOF).

Protocol for PEGylation, standard “HC” method using NHS-activated 20 kDalinear PEG:

1) The protein was evaluated for the presence of endotoxin. A proteinsolution (0.1 mL) was diluted in 0.9 mL fresh MQ water and tested with ahand-held Charles River apparatus (EndoPTS) for endotoxin at the 0.5EU/mL sensitivity level. If endotoxin was greater than 0.5 EU/mL, thenendotoxin was reduced initially by Mustang E filtration, followed bySterogene Etox resin, and less preferably by further chromatographicpurification. Reduction was limited but sufficiently useful by passageover DEAE FF (Amersham) at pH 7.8.

2) Concentration and buffer exchange of protein. The protein wasconcentrated to greater than 25 mg/mL but less than or equal to 75 mg/mLand buffer exchanged to 50 mM KPO₄, pH 8.5. If a spin filter was used toprepare this concentration, the filter was first tested for endotoxin byspinning at reduced speed and time (3000 rpm, 3 minutes) with bufferalone, then testing the retained buffer for endotoxin in the same way asthe protein in step 1. The buffer batch record/recipe for 50 mM KPO4, pH8.5 consisted of water (QS to 1 L), potassium phosphate dibasic (8.4913g/L of 48.75 mM), and potassium phosphate monobasic (0.17011 g/L of 1.25mM). The solution was filtered through a 0.2 μm filter and stored atroom temperature. The concentrated product was slowly filtered (1-2mL/min) through a Mustang E filter acrodisc. A sample diluted andblanked with sterile TBS, pH 7.5 was measured at A280 to determineprotein concentration. The extinction coefficient was 0.83 for NpPAL and0.75 for AvPAL.

3) PEGylation of NpPAL and AvPAL. PEG normally stored at −80° C. waswarmed to room temperature. KPO4 buffer was added to PEG to resuspend byvortexing at maximum speed, and shaking tube hard in hand to ensure alllarge chunks were suspended. The protein was added to the well-suspendedPEG solution within one minute of having first wetted the PEG and mixedby very gentle inversion. Tubes wrapped in aluminum foil were placed onthe axis of a rocker and rocked very gently at room temperature for 3hours. The tubes were filled with TBS (pH 7.5) and sterile filtered. Thesuspensions were either formulated immediately or stored at 4° C. untilready for formulation.

4) Formulation. The formulation buffer recipe/batch record consisted ofwater (QS to 1 L), Tris-Base (3.2 mM), Tris-HCl (16.8 mM), and sodiumchloride; the buffer solution was filtered through a 0.2 μm filter andstored at room temperature. The buffer solution was subjected totangential flow filtration using a Vivaflow 50 (smaller lots) orVivaflow 200 (larger lots) with a 100 MWCO regenerated cellulosemembrane. The solution was flushed with MQ water, 0.1 N NaOH, and 200 mLwater again. The solution was equilibrated with TBS, pH 7.5 at 50 mL/mincross-flow. The pH of the permeate was determined to ensure a pH of 7.5.

The solution was buffer exchanged by first diluting with TBSapproximately 3-fold and returning to original volume at least fourtimes. Cross-flow was typically 180-200 mL/min for both Vivaflow 50 and200.

The final product was filtered through Mustang E. The presence ofendotoxin was evaluated after diluting 0.1 mL with 1.9 mL sterile freshwater. If endotoxin was greater than 1 EU/mL, reduction was conductedwith Sterogene Etox gel. Formulated, sterile PEGylated NpPAL or AvPALwere sealed in vials and placed at −70° C. until ready for in vivostudies.

Example 7 Generation of AvPAL Variants (Cysteine Mutants)

Amino acid substitutions were made in the AvPAL polypeptide to reduceaggregation that occurs in bacterially expressed, recombinant proteins.Protein aggregation may reduce enzyme activity and/or increaseimmunogenicity in vivo. One such form of aggregation occurs as a resultof formation of inter-chain disulfide bonds. To minimize thispossibility, various AvPAL cysteine residues, alone or in combination,were replaced with serine residues.

The AvPAL polypeptide has 6 cysteine residues, at positions 64, 235,318, 424, 503 and 565 (SEQ ID NO:4). The following AvPAL single cysteinemutants were generated: AvPAL_C64S (SEQ ID NO:7), AvPAL_C318S (SEQ IDNO:8), AvPAL_C503S (SEQ ID NO:9), and AvPAL_C565S (SEQ ID NO:10). AnAvPAL double cysteine mutant, AvPAL_S565SC503S (SEQ ID NO:11), was alsogenerated. FIG. 5A-5E shows the amino acid sequences of these AvPALcysteine mutants.

Cloning

The AvPAL gene was amplified from Anabaena variabilis genomic DNA (ATCC29413-U, Qiagen DNeasy Kit) with forward primer AvarPALfor(5′-CACTGTCATATGAAGACACTATCTCAAGCACAAAG-3′) (SEQ ID NO:18) and reverseprimer AvarPALrev (5′-CACTGTCTCGAGATGCAAGCAGGGTAAGATATCTTG-3′) (SEQ IDNO:19). The resulting PCR product was treated with Taq and then ligatedinto pCR2.1 TOPO TA (Invitrogen). The resulting plasmid was named 1p40.

A 5′ NheI site was added and an internal NheI site was removed bySOE-PCR. The upstream AvPAL fragment was amplified from 1p40 withforward primer N-Nhe-AvPAL(5′-CACTGTGCTAGCATGAAGACACTATCTCAAGCACAAAG-3′) (SEQ ID NO:20) andreverse primer Nhe-AvPALrev(5′-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3′) (SEQ IDNO:21), and the downstream AvPAL fragment was amplified from 1p40 withforward primer Nhe-AvPALfor(5′-CCACTAATTGATGTTGATAACCAAGCCAGCTATCATGGAGGAAATTTCC-3′) (SEQ ID NO:22)and reverse primer AvPALrev-r(5′-ACAGTGGCGGCCGCTTAATGCAAGCAGGGTAAGATATCTTG-3′) (SEQ ID NO:23). In asingle PCR reaction, the two PCR products were annealed and extendedwith DNA polymerase to produce the full-length AvPAL gene, and thenamplified with primers N-Nhe-AvPAL and AvPALrev-r. The resulting PCRproduct was digested with NheI, blunted with Klenow, digested with NotI,and ligated into the pET28a+ vector (prepared by digestion with NdeI,blunting with Klenow, and digestion with NotI). The resulting plasmidwas named 3p86-23.

New restriction sites were added by PCR. AvPAL was amplified fromplasmid 3p86-23 with forward primer AvEcoRIfor(5′-ACTGTGAATTCATGAAGACACTATCTCAAGCACAAAG-3′) (SEQ ID NO:24) and reverseprimer AvSmaIrev (5′-CACTGTCCCGGGTTAATGCAAGCAGGGTAAGATATCT-3′) (SEQ IDNO:25). The resulting PCR product was digested with EcoRI and SmaI andligated into EcoRI- and SmaI-digested pIBX7 vector. The resultingplasmid was named 7p56 Av3.

Cysteine Mutants

Two cysteine codons in the AvPAL gene, corresponding to positions 503and 565 of the AvPAL polypeptide, were substituted with serine codons bysite-directed mutagenesis (QuickChange XL II, Stratagene). The cysteinecodon at position 503 was changed to a serine codon in plasmid 7p56 Av3by PCR with forward primer Av_C503S(5′-GTCATTACGATGCACGCGCCTCTCTATCACCTGCAACTGAG-3′) (SEQ ID NO:26) andreverse primer Av_C503Srev(5′-CTCAGTTGCAGGTGATAGAGAGGCGCGTGCATCGTAATGAC-3′) (SEQ ID NO:27). Theserine codon is underlined and the G to C mutation in the coding strand(C to G mutation in the non-coding strand) is indicated in bold. Theresulting plasmid was named j282. The cysteine codon at position 565 waschanged to a serine codon in plasmid j282 with forward primer Av_C565S(5′-CAGTTCAAGATATCTTACCCTCCTTGCATTAACCCGGGCTGC-3′) (SEQ ID NO:28) andreverse primer Av_C565Srev(5′-GCAGCCCGGGTTAATGCAAGGAGGGTAAGATATCTTGAACTG-3′) (SEQ ID NO:29). Theserine codon is underlined and the G to C mutation in the coding strand(C to G mutation in the non-coding strand) is indicated in bold. Theresulting plasmid was named j298a.

Cysteine codons in the AvPAL gene at positions 64, 318 and 565 of theAvPAL polypeptide were similarly substituted with serine codons usingthe following primer pairs: C64S, forward primer Av_C64S(5′-GCAGGGTATTCAGGCATCTTCTGATTACATTAATAATGCTGTTG-3′) (SEQ ID NO:30) andreverse primer Av_C64Srev(5′-CAACAGCATTATTAATGTAATCAGAAGATGCCTGAATACCCTGC-3′) (SEQ ID NO:31);C318S, forward primer Av_C318S(5′-CAAGATCGTTACTCACTCCGATCCCTTCCCCAGTATTTGGGGC-3′) (SEQ ID NO:32) andreverse primer Av_C318Srev(5′-GCCCCAAATACTGGGGAAGGGATCGGAGTGAGTAACGATCTTG-3′) (SEQ ID NO:33); andC565S, forward primer Av_C565S (SEQ ID NO:28) and reverse primerAv_C565Srev (SEQ ID NO:29). The serine codons are underlined, and the Gto C mutations in the coding strands and the C to G mutations in thenon-coding strands are indicated in bold.

Example 8 In Vitro Enzyme Activity of AvPAL Variants (Cysteine Mutants)

The purpose of this study was to determine the effect of serinesubstitution of the various cysteine residues in the AvPAL polypeptideon in vitro phenylalanine ammonia-lyase (PAL) enzyme activity.

AvPAL variants (i.e., cysteine mutants) were cloned as described inEXAMPLE 7. The AvPAL cysteine mutant expression plasmids weretransformed into bacteria and the AvPAL cysteine mutant polypeptideswere expressed as described in EXAMPLE 1 and purified as described inEXAMPLE 2.

The wild-type (WT) AvPAL and AvPAL cysteine mutants were tested for invitro PAL enzyme activity as described in EXAMPLE 3. Table 1 shows thatcompared to unpegylated WT AvPAL, the in vitro PAL specific activity ofthe purified, unpegylated AvPAL cysteine mutant proteins was reduced byserine substitution of the cysteine residue at position 64 (AvPAL_C64S),but was not adversely affected by serine substitution of the cysteineresidues at either of positions 503 or 565, or at both positions 503 and565 (AvPAL_C503S, AvPAL_C565S, and AvPAL_C565SC503S, respectively).

TABLE 1 Specific Activity of AvPAL Cysteine Mutants AvPAL ProteinPEGylation Specific Activity (U/mg) WT AvPAL − 1.7 AvPAL_C503S − 1.9AvPAL_C64S − 1.3 AvPAL_C565S E1 − 2.0 AvPAL_C565S E2 − 2.1AvPAL_C565SC503S − 2.2 WT AvPAL + 1.1 AvPAL_C565SC503S + 1.1

To determine whether the introduction of the serine residues had anyeffect on enzymatic activity of pegylated AvPAL proteins, the WT AvPALand double cysteine mutant, AvPAL_C565SC503S, were pegylated asdescribed in EXAMPLE 6. Table 1 shows that the in vitro PAL specificactivity of the pegylated AvPAL protein was not adversely affected byserine substitution of the cysteine residues at both positions 503 and565.

Example 9 In Vitro Biochemical Characterization of AvPAL Variants(Cysteine Mutants)

The purpose of this study was to determine the effect of serinesubstitution of the various cysteine residues in the AvPAL polypeptideon: (1) accelerated stability; (2) aggregate formation; and (3)site-specific pegylation.

Accelerated Stability

The effect of serine substitution of cysteine residues in AvPAL on invitro stability was determined by storing the purified AvPAL cysteinemutants, either pegylated or unpegylated, for various time periods at37° C., and then measuring the in vitro PAL specific activity of theseproteins as described in EXAMPLE 3.

Wild-type AvPAL and AvPAL cysteine mutants, either upegylated orpegylated, were prepared as described in EXAMPLE 8.

As shown in FIG. 6A, the specific activities of the unpegylated AvPALproteins were stable for at least 5 days at 37° C., and were notadversely affected by serine substitution of the cysteine residues atposition 565, or at both positions 503 and 565. Similarly, as shown inFIG. 6B, the specific activities of the pegylated AvPAL proteins werestable for at least 6 days at 37° C. The single cysteine AvPAL mutant,AvPAL_C565S, showed somewhat reduced stability compared to wild-typeAvPAL and the double cysteine AvPAL mutant, AvPAL_C565SC503S, after 6days at 37° C.

Aggregate Formation

The effect of serine substitution of cysteine residues in AvPAL onformation of protein aggregates in solution was determined by separatingthe purified, unpegylated wild-type AvPAL and AvPAL cysteine mutants byeither denaturing and native gel electrophoresis or by SEC-HPLC.

The purified AvPAL preparations were separated by gel electrophoresisunder either denaturing conditions (4-12% NuPAGE Bis-Tris) or nativeconditions (8% Tris-Gly, pH 8.3). The separated AvPAL proteins werestained with Coomassie Blue.

The purified AvPAL preparations were separated by SEC-HPLC. AvPALproteins were loaded onto a TSK gel column (G3000SWxl, 7.8mm×30 cm, 5(Tosoh Bioscience, LLC)) in 20 mM Na-phosphate, 300 mM NaCl, pH 6.9, andeluted at a flow rate of 0.5 mL/min. The separated AvPAL proteins wereanalyzed on an Agilent series 1100 spectrometer.

Aggregates were present in the wild-type AvPAL preparation and in theAvPAL_C503S and AvPAL_C64S preparations, but not in the AvPAL_C565S andAvPAL_C565SC503S preparations, as judged by either gel electrophoresis(FIG. 7A) or SEC-HPLC (FIG. 7B).

Site-Specific Pegylation

The effect of serine substitution of cysteine residues in AvPAL onsite-specific pegylation was determined by pegylating the wild-typeAvPAL and double cysteine mutant AvPAL_C503SC565S as described inEXAMPLE 6, and then comparing the relative pegylation at the AvPALlysine residues: K2, K10, K32, K115, K145, K195, K301, K335, K413, K419,K493, K494 and K522.

Approximately 100 μg (10 μL at 10 μg/μL) of unpegylated or pegylatedAvPAL proteins were denatured in 8 M urea. The denatured proteins werethen digested in a 100 μL reaction volume with trypsin in 0.8 M urea atpH 8.2 overnight (˜20 hours) at 37° C. The trypsin-digested proteinswere reduced by treatment with 1 μL of 1 M DTT for 1 hour at 37° C.,followed by quenching with 3μL 15% TFA. Digested proteins were separatedon a C18 reverse-phase column. Percent pegylation of each of thepegylated AvPAL peptides was calculated by subtractive peptide mappingof the corresponding unpegylated peptide.

As shown in FIG. 8, at a ratio of AvPAL protein:PEG of 1:3, there was nostriking difference in the percent pegylation of any of the lysine (K)residues with the possible exception of K419, in which the percentpegylation of the double cysteine mutant C565SC503S was lower comparedto wild-type AvPAL. However, the results obtained using the doublecysteine mutant at increasing AvPAL protein:PEG ratios, in which nodose-response relationship was observed, taken together with therelatively small percent pegylation, indicates that the observeddifferences at K1419 are not likely to be meaningful. Thus, serinesubstitution of cysteine residues at positions 503 and 565 does notappear to affect site-specific pegylation of AvPAL.

Example 10 Mechanism of Aggregation of AvPAL Proteins

Studies were performed to investigate the mechanism of aggregation ofbacterially expressed AvPAL proteins.

Concentrating the purified AvPAL preparations, and incubating theconcentrated protein solutions for 2 hours at 37° C., acceleratedaggregation of purified AvPAL proteins in solution. Aggregation wasdetected by separating the AvPAL proteins by SEC-HPLC. To determinewhether disulfide cross-linking was responsible for the aggregation, 50mM dithiothreitol (DTT) was added to the concentrated protein solution,followed by incubation for 2 hours at 37° C.

AvPAL proteins expressed in bacteria were purified as described inEXAMPLE 2, and concentrated using a spin filter (Millipore Biomax −10KNMWL). Proteins were spun at about 15,000 g for a few minutes in anEppendorf Centrifuge 5415C. For cysteine mutants that tend to aggregate(e.g., AvPAL_C503S and AvPAL_C64S), proteins were concentrated to about20 mg/mL and incubated for 2 hours at 37° C. For cysteine mutants thatare resistant to aggregation (e.g., AvPAL_C565S and AvPAL_C565SC503S),proteins were concentrated to about 40 mg/mL and incubated for 2 hoursat 37° C.

As shown in Table 2, preparations of purified AvPAL cysteine mutantsAvPAL_C64S and AvPAL_C503S formed aggregates upon incubation for 2 hoursat 37° C. As expected, this aggregation was exacerbated when the AvPALproteins were concentrated prior to incubation for 2 hours at 37° C. Theaggregation could be blocked by exposure of the concentrated proteins toDTT, indicating that the aggregation is due to disulfide cross-linking.In contrast, the preparations of purified AvPAL cysteine mutantsAvPAL_C565S and AvPAL_C565SC503S did not form aggregates upon incubationfor 2 hours at 37° C., indicating that the cysteine residue at position565 is involved in aggregation of AvPAL via disulfide cross-linking.

TABLE 2 Disulfide Cross-link Related Aggregation of AvPAL CysteineMutants Aggregate AvPAL Protein Treatment Formation AvPAL_C503S 37° C./2h + AvPAL_C64S 37° C./2 h + AvPAL_C565S E1 37° C./2 h − AvPAL_C565S E237° C./2 h − AvPAL_C565SC503S 37° C./2 h − AvPAL_C503S Concentrate + 37°C./2 h ++ AvPAL_C64S Concentrate + 37° C./2 h ++ AvPAL_C565S E1Concentrate + 37° C./2 h − AvPAL_C565S E2 Concentrate + 37° C./2 h −AvPAL_C565SC503S Concentrate + 37° C./2 h − AvPAL_C503S Conc. + DTT +37° C./2 h − AvPAL_C64S Conc. + DTT + 37° C./2 h − AvPAL_C565S E1Conc. + DTT + 37° C./2 h − AvPAL_C565S E2 Conc. + DTT + 37° C./2 h −AvPAL_C565SC503S Conc. + DTT + 37° C./2 h −

To determine which cysteine residues exist as free sulfhydryls, apurified AvPAL preparation was denatured in the presence of 8 M urea,alkylated by iodoacetamide, digested with trypsin, and analyzed byLC/MS. All of the AvPAL cysteine residues were labeled by iodoacetamide,indicating that all of the cysteine residues of bacterially expressedAvPAL exist as free sulfhydryls (data not shown).

To determine which cysteine residues are present on the surface of thenative protein, a purified AvPAL preparation was first treated withN-ethylmaleimide (NEM), then denatured in the presence of 8 M urea,alkylated by iodoacetamide, digested with trypsin, and analyzed byLC/MS. The cysteine residues at positions 235 and 424 were not alkylatedby NEM, and the cysteine residue at position 318 was only partiallyalkylated by NEM, indicating that the cysteine residues at positions 64,503 and 565 are on the surface of native AvPAL and the cysteine residueat position 318 is partially exposed on the surface of native AvPAL(data not shown).

To determine which cysteine residues are involved in the inter-chaindisulfide cross-linking, 67 μL of a 0.7 mg/mL solution of purified,unpegylated wild-type AvPAL preparation was denatured and alkylated in 8M urea containing 20 mM iodoacetamide for 1 hour at 37° C., and thendigested in a 100 μL reaction volume with trypsin at pH 8.2 overnight(˜17.5 hours) at 25° C. The trypsin-digested proteins were separated andanalyzed by mass spectrometry, in which peptides corresponding to thepredicted disulfide pairs were identified and quantitated as total ioncounts (TIC).

Table 3 shows that disulfide pairs were detected for C503-C503,C503-0565, C565-C318 and C565-0565. The cysteine residues at position565, and to a lesser extent at position 503, were found in disulfidepairs in the purified AvPAL preparation.

TABLE 3 Aggregate Disulfide Pairs Disulfide Pair Results (TIC/1000)C64-C318  n.d.^(#) C64-C64 n.d. C64-C503 n.d. C64-C565 n.d. C503-C318n.d. C503-C503 11 C503-C565 112 C565-C318 13 C565-C565 37 C318-C318 n.d.^(#)not detected

Studies were performed to determine whether additional mechanismsbesides disulfide cross-linking might be involved in AvPAL proteinaggregation.

Purified AvPAL preparations were incubated with either 0.05% Tween or 10mM EDTA, and then separated by SEC-HPLC as described in EXAMPLE 9. Tweenreduces protein aggregation due to hydrophobic interactions, and EDTAreduces protein aggregation due to the presence of divalent cations. Asshown in FIG. 9, exposure to 0.05% Tween or 10 mM EDTA had no effect onAvPAL protein aggregation. The additional peak at 10 minutes in the 10mM EDTA treated AvPAL is due to absorbance of EDTA at 210 nm.

To further investigate the role of disulfide cross-linking in AvPALprotein aggregation, purified AvPAL was reduced by treatment with DTTand then desalted prior to separation by SEC-HPLC. As shown in FIG. 10A,AvPAL protein aggregation was minimized by treatment with DTT, andaggregates re-formed following incubation for 18 hours at 37° C. Incontrast, as shown in FIG. 10B, aggregates did not re-form once theAvPAL surface cysteines were modified (i.e., alkylated) by treatmentwith N-methylmaleimide (NEM) after DTT exposure, but before desaltingand incubation for 18 hours at 37° C.

Based on the above, aggregation of bacterially expressed AvPAL appearsto be due solely to formation of inter-chain disulfide bonds, and notdue to hydrophobic effects or presence of divalent cations. The cysteineresidues at positions 565 and 503 are involved in formation ofinter-chain disulfide bonds in AvPAL preparations.

Example 11 Liquid Formulations of PEGylated Forms of AvPAL Variants(Cysteine Mutants)

Studies were performed to investigate the effect of various excipients,e.g., stabilizers, on the accelerated stability of a PEGylated form ofan AvPAL polypeptide variant (e.g., with serine substitution of thecysteine residues at positions 503 and 565) in formulations of theinvention.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was preparedas described in EXAMPLE 7.

Accelerated stability of different formulations of pegylatedAvPAL_C565SC503S was determined using an in vitro activity assay, eithera cuvette assay or a plate assay. For the cuvette assay, purifiedpegylated AvPAL_C565SC503S was diluted in TBS dilution buffer and thenadded to an assay buffer containing 22.5 mM phenylalanine (Phe), 100 mMTris-HCl, pH 8.5. After incubation for 2 minutes at 30° C., the amountof trans-cinnamic acid (t-CA) released was measured by absorbance at 290nm. For the plate assay, purified pegylated AvPAL_C565SC503S was dilutedin TBS dilution buffer plus BSA/Phe/Brij and then added to an assaybuffer containing 22.5 mM Phe, 100 mM Tris-HCl, pH 8.5. After incubationfor 10-20 minutes at 30° C., the amount of trans-cinnamic acid (t-CA)released was measured by absorbance at 290 nm. One IU of PAL activity isequal to 1 μMol TCA/min.

In a first accelerated stability study, the effect of pH on stability ofthe pegylated double cysteine mutant AvPAL AvPAL_C565SC503S wasevaluated. Purified pegylated AvPAL_C565SC503S was pre-formulated in 10mM buffer and 140 mM NaCl at various pH, from 4 to 9. Buffers tested:citrate (pH 4), acetate (pH 5), histidine (pH 6). phosphate (pH 7), Tris(pH 7.5, pH 8) and arginine (pH 9). After storing the enzymeformulations for up to 30 days at 4° C., 25° C. or 37° C., in vitroactivity was measured. A total loss of PAL enzyme activity was observedat pH 4. A pH range from 7 to 8 was chosen for further evaluation.

In a second accelerated stability study, the effect of pH and a varietyof excipients on stability of the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S was evaluated. Purified pegylated AvPAL_C565SC503S waspre-formulated in 10 mM Tris and 140 mM NaCl at pH 7, 7.5 or 8.0 in theabsence or presence of 0.5% EDTA, 0.5% EDTA plus 0.5% ascorbic acid or0.5% EDTA plus 5 mM methionine (Met). After storing the enzymeformulations for up to 60 days at 4° C., 25° C. or 37° C., in vitroactivity was measured. pH 7.0 and 7.5 appeared equivalent in maintainingenzyme activity, EDTA had little or no effect on enzyme activity, andthe anti-oxidants ascorbic acid and methionine negatively affectedenzyme activity.

In the same accelerated stability study, the effect of pegylation of theAvPAL double cysteine mutant AvPAL_C565SC503S was evaluated. The rate ofloss of enzyme activity was similar between unpegylated and pegylatedAvPAL_C565SC503S.

In a third accelerated stability study, the effect of enzyme substrateand product as excipient on stability of the pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S was evaluated. Purified pegylatedAvPAL_C565SC503S at approximately 12 mg/mL (0.2 mM) was pre-formulatedin 10 mM Tris and 140 mM NaCl at pH 7.5 in the absence or presence of 1mM Phe (substrate at 5 moles per mole active site), 2 mM TCA (product at10 moles per mole active site) or 0.05% Tween 80 (a surfactant). Afterstoring the enzyme formulations for various times at 4° C., 25° C. or37° C., in vitro activity was measured weekly. Both Phe and t-CAsignificantly increased stability of the enzyme, whereas Tween had noeffect on enzyme stability.

A summary of the accelerated stability studies 1, 2 and 3 is shown inFIG. 11.

In a fourth accelerated stability study, the effect of Phe and t-CA atlow concentrations as excipient on stability of the pegylated AvPALdouble cysteine mutant AvPAL_C565SC503S was evaluated. Purifiedpegylated AvPAL_C565SC503S at approximately 12 mg/mL (0.2 mM) waspre-formulated in 10 mM Tris and 140 mM NaCl at pH 7.5 in the absence orpresence of 0.4 mM Phe (substrate at 2 moles per mole active site) or0.4 mM TCA (product at 2 moles per mole active site). After storing theenzyme formulations for various times at 4° C., 25° C. or 37° C., invitro activity was measured weekly. Both Phe and t-CA at lowconcentration were effective at stabilizing enzyme activity.

In a fifth accelerated stability study, the effect of a weak enzymesubstrate, tyrosine (Tyr), as excipient on stability of the pegylatedAvPAL double cysteine mutant AvPAL_C565SC503S was evaluated. Purifiedpegylated AvPAL_C565SC503S at approximately 12 mg/mL (0.2 mM) waspre-formulated in 10 mM Tris and 140 mM NaCl at pH 7.5 in the absence orpresence of 1 or 5 mM Tyr (substrate at 5 or 25 moles per mole activesite, respectively). After storing the enzyme formulations for varioustimes at 4° C., 25° C. or 37° C., in vitro activity was measured weekly.Tyr had a minimal, non-dose dependent stabilizing effect on enzymeactivity (FIG. 12).

In a sixth accelerated stability study, the effect of nucleophilicscavengers as excipient on stability of the pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S was evaluated. Purified pegylatedAvPAL_C565SC503S at approximately 20 mg/mL (0.33 mM) was pre-formulatedin 10 mM Tris and 140 mM NaCl at pH 7.5 in the absence or presence of 1Phe (substrate at 3 moles per mole active site), 2 mM nucleophilicscavenger (either benzoic acid or pyridoxamine at 6 moles per moleactive site), or both 1 mM Phe and 2 mM nucleophilic scavenger. Afterstoring the enzyme formulations for various times at 4° C. or 37° C., invitro activity was measured weekly. Benzoic acid, but not pyridoxamine,was effective at stabilizing enzyme activity (FIG. 13A). There was noadditive effect of Phe and benzoic acid, suggesting a similarstabilizing mechanism.

The stabilizing effects of benzoic acid and t-CA suggest that theyfunction as structural analogs of Phe (see FIG. 13B).

The data from the six accelerated stability studies were combined inorder to predict the effective shelf-life of the pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S in various formulations. Shelf-life wasdetermined as follows: (1) determining the rate of activity decay(k_(decay)), which followed first order kinetics, for each formulationcondition; (2) plotting the ln(k_(decay)) v. 1/Temperature (° K.); (3)determining the Ea (ΔG_(decay)) required for activity decay for a givenformulation condition; (4) extrapolating the k_(decay) at 4° C. usingthe calculated Ea and the observed k_(decay) at a given temperature; and(5) detetrmining the shelf life (T₉₀), which is the time in whichspecific enzyme activity has dropped by ≧10% at 4° C.

Table 4 shows that Phe and t-CA greatly enhances the predictedshelf-life of the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S.

TABLE 4 Predicted Shelf-Life T₉₀ (in Weeks) of Pegylated Double CysteineMutant AvPAL_C565SC503S with Various Excipients Excipient 42° C. 37° C.25° C. 4° C.* 4° C. (Observed) None (TBS) 0.67 0.8 2.1 12.9 ~9-13 Phe1.63 2.2 9.1 85 >20 t-CA ND 2.0 7.1 85.8 >20 *Numbers are estimatesbased on data from up to 6 different experiments.

In summary, the above preformulation studies indicate that the pHoptimum for the pegylated AvPAL double cysteine mutant AvPAL_C565SC503Sis 7 to 7.5. The presence of anti-oxidants result in a drastic loss ofenzyme activity. Both phenylalanine (Phe) and transcinnamic acid (t-CA)increase the stability of rAvPAL-PEG by 50% or more under acceleratedconditions (25° C. and 37° C.). A 2-fold excess Phe or t-CA perrAvPAL-PEG active site is sufficient to stabilize activity and higherconcentrations appear to have no additional benefit. A weaker PALsubstrate, tyrosine (Tyr), does not appear to stabilize enzyme activity,whereas benzoic acid, stabilizes rAvPAL-PEG activity to a similar degreeas its structural analog, Phe. When combined with Phe, no additionalactivity stabilization is observed with benzoic acid, suggesting acommon mechanism for activity stabilization.

Example 12 Lyophilized Formulations of PEGylated Forms of AvPAL Variants(Cysteine Mutants)

Studies were performed to investigate the effect of various solid (e.g.,lyophilized) formulations on the activity of a PEGylated form of anAvPAL polypeptide variant (e.g., with serine substitution of thecysteine residues at positions 503 and 565) of the invention.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was preparedas described in EXAMPLE 7.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S wasformulated as follows: (F1) 10 mg/mL AvPAL_C565SC503S, 10 mM Tris, pH7.5; (F2) 10 mg/mL AvPAL_C565SC503S, 10 mM Tris, pH 7.5, 25 mg/mLmannitol; or (F3) 10 mg/mL AvPAL_C565SC503S, 10 mM Tris, pH 7.5, 20mg/mL mannitol, 5 mg/mL sucrose. After formulation, the PAL enzymeactivity of each was 1.7 to 1.8 U/mg. After lyophilization, theformulations were stored for up to 26 at 4° C., and then resuspended infresh, sterile-filtered MilliQ water. The PAL enzyme activities weredetermined as described in EXAMPLE 11. Table 5 shows that there appearedto be no loss of activity upon lyophilization, storage or resuspensionof the various AvPAL_C565SC503S formulations.

TABLE 5 Specific Activity of Pegylated Double Cysteine MutantAvPAL_C565SC503S Upon Lyophilized Formulation (LF) After LF + After LF +After LF + 11 days/ 26 days/ LF Before LF After LF 5 days/4° C. 4° C. 4°C. F1 1.78 +/− 0.04 1.60 1.59 1.71 1.48 F2 1.72 +/− 0.01 1.67 1.62 1.681.72 F3 1.65 +/− 0.09 1.66 1.73 1.76 1.59

Example 13 Toxicity/Pharmacokinetic Studies of PEGylated Forms of AvPALVariants (Cysteine Mutants) in Cynomolgus Monkeys and Rats

Toxicity/pharmacokinetic studies were performed to determine the effectof administration of a single dose of a PEGylated form of an AvPALpolypeptide variant (e.g., with serine substitution of the cysteineresidues at positions 503 and 565) in Cynomolgus monkeys and in rats.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was preparedas described in EXAMPLE 7.

Cynomolgus Monkey Toxicity/Pharmacokinetic Study

This study used four (4) groups of monkeys, each with three males andthree females. Group 1 received placebo (mL/kg); and Groups 2, 3 and 4received a single subcutaneous injection of pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S in solution at 4, 12 and 60 mg/kg,respectively. Plasma samples were collected from the monkeys pre-dose,and at various times post-dose, from 3 to 504 hours. The 60 mg/kg dosewas found to be toxic to the monkeys, so the Group 4 portion of thisstudy was terminated.

FIG. 14A shows the concentration of pegylated AvPAL double cysteinemutant AvPAL C565SC503S in the plasma at various times after a singlesubcutaneous injection at 4 and 12 mg/kg. The data shows monophasicelimination of the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S. A single compartment model with 1^(st) orderabsorption appears to describe the plasma profile of the pegylated AvPALdouble cysteine mutant AvPAL_C565SC503S after a single subcutaneousinjection.

FIG. 14B shows the concentrations of phenylalanine (Phe) and pegylatedAvPAL double cysteine mutant AvPAL_C565SC503S in the plasma at varioustimes after a single subcutaneous injection at 4 mg/kg. At this dose,the plasma Phe concentration was reduced to below the limit ofquantitation in the GC/MS assay within 24 hours, and the drop in plasmaPhe was sustained over 10 days.

Rat Toxicity/Pharmacokinetic Study

This study used eight (8) groups of rats, with 3 males and 3 females inthe placebo groups, and 6 males and 6 females in the test groups. Groups1 and 5 received single intravenous and subcutaneous injections ofplacebo. Groups 2, 3 and 4 received single intravenous injections ofpegylated AvPAL double cysteine mutant AvPAL_C565SC503S at 1, 5 and 25mg/kg, respectively. Groups 6, 7 and 8 received single subcutaneousinjections of pegylated AvPAL double cysteine mutant AvPAL_C565SC503S at10, 25 and 250 mg/kg, respectively. Blood samples were collected fromthe rats pre-dose, and at various times post-dose, from 1 to 360 hours.At each collection time, blood was collected from 3 rats in each group.No toxicity was observed in the rats in this study.

FIG. 15A shows the concentration of pegylated AvPAL double cysteinemutant AvPAL_C565SC503S in the plasma at various times after a singleintravenous injection at 1, 5 and 25 mg/kg. The data shows monophasicelimination of the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S from the plasma after a single intravenous injection.

FIG. 15B shows the concentration of pegylated AvPAL double cysteinemutant AvPAL_C565SC503S in the plasma at various times after a singlesubcutaneous injection at 10, 25 and 250 mg/kg. A single compartmentmodel with first order absorption appears to describe the plasma profileof the pegylated AvPAL double cysteine mutant AvPAL_C565SC503S after asingle subcutaneous injection.

Table 6 shows pharmacokinetic parameters of the pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S after a single intravenous orsubcutaneous injection.

TABLE 6 Pharmacokinetic Parameters of Pegylated Double Cysteine MutantAvPAL_C565SC503S After a Single Intravenous or Subcutaneous Dose DoseAUC_(0-∞) C_(max) T_(max) t_(1/2)* F Route (mg/kg) (ng-hr/mL) (ng/mL)(hr) (hr) (%) Intravenous 1 657131 12600 4.5 27.9 — 5 3579327 87667 239.1 — 25 10860907 202238 9.0 30.4 — Subcutaneous 10 1304016 16674 18.046.9 19.7 25 2290754 29260 42.0 21.0 12.5^(#) 250 37254683 225200 72.062.8 34.0 *For the subcutaneous route of administration, terminalt_(1/2) is longer than intravenous; this may be due to a slower rate ofabsorption from subcutaneous tissues than the rate of elimination (sothat the t_(1/2) observed is actually absorption). ^(#)Bioavailabilityusing intravenous AUC data at 25 mg/kg is 21.5%.

There appeared to be no gender difference in this pharmacokinetic study.The AUC_(inf) and C_(max) were roughly proportional with dose for boththe intravenous and subcutaneous routes of administration.

Multiple Dose Toxicity Studies in Rats and Cynomolgus Monkeys

The safety of pegylated AvPAL double cysteine mutant AvPAL_C565SC503Swas evaluated in repeat-dose toxicity studies in rats and Cynomolgusmonkeys.

Rats administered up to 25 mg/kg pegylated AvPAL double cysteine mutantAvPAL_C565SC503S twice weekly, subcutaneously over 28 days exhibited notoxicity.

Cynomolgus monkeys administered up to doses of 1 mg/kg pegylated AvPALdouble cysteine mutant AvPAL_C565SC503S twice weekly, subcutaneouslyover 28 days exhibited no significant toxicity. A dose dependentdecrease in plasma Phe levels was observed after the first dose;however, after the seventh dose, plasma Phe levels returned to baselinein all dose groups, indicating a possible antibody response toward theadministered enzyme. Minimal anti-AvPAL_C565SC503S IgG titers wereobserved in most 1 mg/kg treated animals at day 28. No IgM titers wereobserved in any animal in the study at day 28.

Example 14 Effects of AvPAL Variants (Cysteine Mutants) on Tumor Cellsin Culture

Studies were performed to investigate the effect a PEGylated form of anAvPAL polypeptide variant (e.g., with serine substitution of thecysteine residues at positions 503 and 565) on the proliferation oftumor cells grown in culture in vitro.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was preparedas described in EXAMPLE 7.

The proliferation of tumor cells in vitro was measured using a propidiumiodide fluorescence assay as described in Dengler, et al., Anti-CancerDrugs 6:522-532 (1995).

Hematological Tumors

A panel of twenty-four (24) hematological tumor cell lines, including 14leukemias, 5 lymphomas and 5 myelomas, were evaluated for the effect ofpegylated double cysteine mutant AvPAL_C565SC503S on cell proliferationin vitro.

The hematological tumor cell lines were seeded into culture plates at5,000 cells/well on Day 0. On Day 1, pegylated double cysteine mutantAvPAL_C565SC503S was added to the cultures at various concentrations,from 0.01 to 100 μg/mL. On Day 5, cells were harvested and DNA contentwas measured by propidium iodide staining. The IC₅₀, IC₇₀ and IC₉₀ weredetermined. These experiments were performed twice or three times foreach hematological tumor cell line.

Table 7 shows that pegylated double cysteine mutant AvPAL_C565SC503S waseffective in inhibiting in vitro proliferation, as measured by propidiumiodide staining, of several hematological tumor cell lines.

TABLE 7 Inhibition of Propidium Iodide Staining of Hematological TumorLines In Vitro by Pegylated Double Cysteine Mutant AvPAL_C565SC503STumor IC₅₀ IC₇₀ Line Cell Type μg/mL μg/mL CCRF CEM ALL - T CellLymphoma 1 >100 >100 >100 >100 >100 EM2CML >100 >100 >100 >100 >100 >100 HL-60 APL 0.904 >100 >100 >10046.41 >100 JURKAT Human T Cell Leukemia 0.38 2.928 14.125 >10010.000 >100 JURLMK1 CML 0.766 >100 10 >100 3.162 >100 K562 CML 0.70159.948 >100 >100 11.659 >100 KCL22 CML 0.9 15.399 >100 >100 1 >100 KG1AML 43.287 >100 >100 >100 >100 >100 MEG01 CML 1.258 >100 >100 >1000.926 >100 MOLT4 ALL - T cell lymphoma 0.326 1.873 1.082 5.298 1.0966.918 Mv411 AML 5.994 74.989 >100 >100 >100 >100 NOMO1 AML 0.304 2.5110.732 8.659 0.863 6.449 OCIAML2 AML 0.261 0.938 >100 >100 7.305 >100PL21 AML >100 >100 >100 >100 >100 >100 HUT78 Lym CTL 6.105 18.27617.782 >100 0.096 >100 L5178Y Mouse T cell Leukemia 6.683 41.595 3.98110 3.019 7.585 MYLA Lym CTL 4.436 >100 5.379 >100 8.171 >100 RAJIBurkitt Lymphoma 0.261 0.938 21.544 >100 2.154 >100 U937 Histio Lymphoma0.803 >100 >100 >100 >100 >100 8226 Myeloma 0.229 0.825 >100 >100 0.6917.742 IM9 Human Lymphoblastic Cells 0.271 1.467 0.295 1.311 0.063 0.188L363 Human Plasma Cell Leukemia 7.943 >100 1.73 15.505 LP1 HumanMultiple Myeloma 0.774 100 0.71 6.309 NCIH929 Human MultipleMyeloma >100 >100 11.288 >100 2.154 >100

Dose-dependent inhibition of cell proliferation, as determined by areduction in propidium iodide staining, by the pegylated double cysteinemutant AvPAL_C565SC503S in two sensitive hematological tumor cells,NOMO1 and IM9, are shown in FIGS. 14A and 21B, respectively. These tumorcell lines had IC₅₀ and IC₇₀ of less than 1.0 and 10.0 μg/mL,respectively. For comparison, asparaginase has an IC₅₀ of 1-10 μg/mL inhuman leukemia cell lines. In general, however, the hematological tumorcell lines were more resistant, as judged by IC₇₀ values, than the solidtumor lines (see below).

Solid Tumors

A panel of thirty-six (36) solid tumor cell lines, including tumorsderived from bladder, brain, colon, stomach, head and neck, lung,breast, ovary, pancreas, prostate, kidney and uterus, were evaluated forthe effect of pegylated double cysteine mutant AvPAL_C565SC503S on cellproliferation in vitro. The solid tumor cell lines were seeded intoculture plates at 5,000 cells/well on Day 0. On Day 1, pegylated doublecysteine mutant AvPAL_C565SC503S was added to the cultures at variousconcentrations, from 0.01 to 100 μg/mL. On Day 5, DNA content wasmeasured by propidium iodide staining. The IC₅₀, IC₇₀ and IC₉₀ weredetermined.

Table 8 shows that pegylated double cysteine mutant AvPAL_C565SC503S waseffective in inhibiting in vitro proliferation, as measured by propidiumiodide staining, of several solid tumor cell lines.

TABLE 8 Inhibition of Propidium Iodide Staining of Solid Tumor Lines InVitro by Pegylated Double Cysteine Mutant AvPAL_C565SC503S Tumor IC₅₀IC₇₀ Line Cell Type μg/mL μg/mL Bladder 1218L ATCC, Freiburg; Urothelial1.1 7.498 Adenocarcinoma T24 Xenograft 0.617 2.154 Brain/CNS 498NLXenograft, Freiburg 0.691 2.154 SF268 NCI 0.59 1.492 Colon HCT116 NCI;Adenocarcinoma, pd 0.316 0.9 HT29 NCI; Adenocarcinoma, pd 0.508 0.94Gastric 251L Xenograft, Freiburg; Adenocarcinoma, pd 2.682 37.275 Headand Neck 536L Xenograft, Freiburg; Hypopharynx 0.606 1.887 CarcinomaLung 1121L Xenograft 0.715 3.548 289L Xenograft, Freiburg;Adenocarcinoma, pd 2.807 23.101 529L Xenograft, Freiburg; Large Cell, du0.539 1.73 629L Xenograft, Freiburg; Adenocarcinoma, pd 0.457 1.467 H460NCI; Large Cell Carcinoma 0.215 0.644 Breast 401NL Xenograft, Freiburg;Pap 1.873 7.564 Adenocarcinoma, wd MCF7 NCI; Mammary Carcinoma 0.5991.623 Melanoma 276L Xenograft 4.124 268.269 394NL Xenograft 0.887 3.856462NL Xenograft 0.954 6.189 514L Xenograft 0.828 4.216 520L Xenograft1.359 6.309 Ovarian 1619L Xenograft, Freiburg; Adenocarcinoma, md 0.3220.688 899L Xenograft, Freiburg; Pap Serous 1.279 6.628 Carcinoma, mdOVCAR3 NCI; Adenocarcinoma, md 1.185 6.528 Pancreatic 1657L Xenograft,Freiburg; Adenocarcinoma, md 1.951 8.619 PANC1 ATCC 0.825 5.179 Prostate22RV1 ATCC; Adenocarcinoma, md 0.87 7.079 DU145 NCI; Adenocarcinoma, md0.622 1.873 LNCAP DSMZ; Adenocarcinoma, md 0.584 0.974 PC3M NCI;Adenocarcinoma, md 0.501 1.274 Pleuramesothelioma 1752L Xenograft,Freiburg; Pleuramesothelioma 1.637 8.483 Renal 1781L Xenograft,Freiburg; Renal Carcinoma 2.371 10 393NL Xenograft, Freiburg;Hypernephroma, wd 0.55 1.995 486L Xenograft 0.859 5.336 944L Xenograft0.71 3.727 Uterine 1138L Xenograft, Freiburg; Carcinosarcoma, wd 0.6211.258

Dose-dependent inhibition of cell proliferation, as determined by areduction of propidium iodide staining, by the pegylated double cysteinemutant AvPAL_C565SC503S in tumor cell lines derived from brain/CNS,colon, lung and prostate cancer is shown in FIG. 17A-D, respectively.

The AvPAL_C565SC503S displayed a selective anti-proliferative activityin this broad panel of solid tumor cell lines, and was particularlypotent (i.e., IC₅₀ between 0.2 and 0.7 μg/mL) in tumor cell linesderived from lung, brain/CNS, colon, prostate and kidney. At least ontumor cell line derived from bladder, head and neck, breast, ovary anduterus were also sensitive to cell killing by AvPAL_C565SC503S. Severalmelanomas were also sensitive to cell killing by AvPAL_C565SC503S.

Example 15 Antitumor Activity of AvPAL Variants (Cysteine Mutants) inNude Mice

Studies are performed to investigate the effect of a PEGylated form ofan AvPAL polypeptide variant (e.g., with serine substitution of thecysteine residues at positions 503 and 565) on the proliferation oftumor cells grown in nude mice in vivo.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S is preparedas described in EXAMPLE 7.

Subcutaneous xenografts of human tumor cells in immunodeficient nude orSCID mice have been successfully used as models for human cancers totest the in vivo efficacy of cancer therapeutic agents as well astargeted cancer therapeutic agents, such as antibodies and toxinconjugates (for review, see Kerbel, Cancer Biol. Ther. 2(4):Suppl.1:S134-S139 (2003)).

The in vivo antitumor activity of the pegylated AvPAL double cysteinemutant AvPAL_C565SC503S can be tested alone or in combination withcancer therapeutic agents or targeted cancer therapeutic agents, or incombination with a phenylalanine-restricted diet, using xenografts ofhuman tumor cells in nude mice.

To establish human tumor xenografts, nude mice are injectedsubcutaneously with about 5×10⁶ human tumor cells in 0.2 mL PBS. Theaverage tumor size increases over time. Human xenograft tumors areexcised from the tumor bearing nude mice and tumor tissue blocks ofapproximately 30 mm³ are prepared. Naive nude mice to be used forevaluating in vivo antitumor activity of pegylated AvPAL double cysteinemutant AvPAL_C565SC503S are each implanted subcutaneously with one tumortissue block. Therapeutic treatment is initiated before tumor initiationor when the average tumor size within a group of nude mice isapproximately 100-150 mm³ (prevention model), and/or after theestablishment of tumors when the average tumor size within a group ofnude mice is above 500 mm³ (treatment model).

In a first step, the dose of pegylated AvPAL double cysteine mutantAvPAL_C565SC503S that will lower plasma phenylalanine (Phe) levels tonear zero is determined. Experiments are performed such as thosedescribed in Examples 7 to 9 and 14 in prior co-pending U.S. patentapplication Ser. No. 11/451,999 filed on Jun. 12, 2006, except nude micerather than ENU2 mice are used. The PAL enzyme dose and the frequency ofadministration are determined in this initial step.

In a second step, the anti-tumor activity of pegylated AvPAL doublecysteine mutant AvPAL_C565SC503S is assessed in various human tumorxenografts derived from patients or cell lines. Tumor models includedifferent cancer types, for example and not for limitation, centralnervous system (CNS), colon, lung, prostate, metastatic melanoma andrenal cancer. Non-comprehensive lists of tumors and tumor cell linesthat can be tested are provided in Tables 7 (hematological tumors) and 8(solid tumors).

To assess the antitumor activity of pegylated AvPAL double cysteinemutant AvPAL_C565SC503S, nude mice bearing different human tumorxenografts subcutaneously are treated with AvPAL_C565SC503S givensubcutaneously at, e.g., three different dose levels, ranging from about5 to 500 mg/kg. This dose may result in a human dose of about 0.1 to 10mg/kg. Antitumor activity is analyzed as tumor volume inhibition and/orabsolute growth delay. The tolerability of the AvPAL_C565SC503S is alsoevaluated as mortality and/or body weight changes.

Each in vivo antitumor study consists of at least four groups, onevehicle control group and at least three prokaryotic PAL enzyme-treatedgroups. The group size will be at least 8 mice, resulting in a total of32 mice receiving subcutaneous tumor implantations. Mice with similarsized tumors (100-150 mm³) will be used for randomization (Day 0).

In the case of an antitumor effect, mice may be monitored for additional2 weeks after termination of prokaryotic PAL enzyme treatment to detecta possible reinitiation of tumor growth. According to regulations foranimal experiments, mice are sacrificed if the tumor diameters exceed1.6 cm.

Tumor diameters are measured twice weekly together with body weight.Tumor volume is calculated according to the formula a*b²/2 (where ‘a’ isthe largest diameter of the tumor and ‘b’ is the perpendicular axis).Relative tumor volumes and body weights are calculated for eachindividual tumor based on the value on Day 0 (the first day of dosing).Treatment starts when the tumors have reached a volume of approximately100-150 mm³. Mice are sacrificed if the tumor volume exceeds 1600 mm³,per regulations for animal studies.

Patient-derived tumors established in serial passage in nude mice canalso be used as test tumors. Typically, these tumors retain importantcharacteristics of the original patient tumor, including histology anddrug sensitivity. For certain tumors, e.g., one CNS and both prostatecancers, cancer cell line-derived tumors are used.

Example 16 Exemplary Prokaryotic PAL Formulations

The following example provides guidance on the parameters to be used toformulate compositions comprising prokaryotic PAL or biologically activefragments, mutant, variants or analogs thereof, which are useful fortreatment of neoplastic disease and cancer. Parameters to be used toformulate prokaryotic PAL compositions of the invention include, but arenot limited to, buffering agents to maintain pH, isotonicity-adjustingagents, absence or presence of stabilizers, and absence or presence ofother excipients, vehicles, diluents, and the like.

In EXAMPLE 11, the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S was formulated at a concentration of about 12-20 mg/mL(about 0.2-0.33 mM). A preferred prokaryotic PAL variant is formulatedat a concentration ranging from about 1 to 50 mg/mL (about 0.016 to 0.8mM), preferably from about 5 to 20 mg/mL (about 0.08 to 0.33 mM), andmore preferably from about 5 to 15 mg/mL (about 0.08 to 0.25 mM). A mostpreferred formulation of the prokaryotic PAL compositions of theinvention has a PAL enzyme concentration of about 10+/−5 mg/mL (about0.16+/−0.08 mM).

In EXAMPLE 11, the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S was formulated in 10 mM Tris-HCl, 140 mM NaCl at pH7.0, 7.5 and 8.0. A preferred buffering agent is Tris-HCl, or itsequivalent, with a concentration ranging from 5 to 50 mM, preferablyfrom 5 to 20 mM, and more preferably from 5 to 15 mM. A most preferredformulation of the prokaryotic PAL compositions of the invention has aTris-HCl buffer concentration of about 10+/−5 mM.

A preferred pH of the pharmaceutical composition is about pH 6.0-8.5,preferably about pH 7.0-8.0, and more preferably about pH 7.0-7.6. Amost preferred formulation of the prokaryotic PAL compositions of theinvention has a pH of about pH 7.3+/−0.3.

A preferred isotonicity-adjusting agent is NaCl, or its equivalent, witha concentration ranging from about 100 to 200 mM, preferably from about130 to 170 mM, and more preferably from about 130 to 150 mM. A mostpreferred formulation of the prokaryotic PAL compositions of theinvention has a NaCl concentration of about 140+/−10 mM.

As shown in EXAMPLE 11, the pegylated AvPAL double cysteine mutantAvPAL_C565SC503S was stabilized in the presence of phenylalanine (Phe),and certain of its structural analogs, including, for example,trans-cinnamic acid (t-CA) and benzoic acid; tyrosine (Tyr) had aminimal stabilizing effect on the PAL enzyme. A preferred stabilizer isPhe, or structural analog thereof, with a range for the stabilizer fromabout 0.1 to 20 moles of stabilizer per mole active site of prokaryoticPAL, preferably from about 0.5 to 10 moles of stabilizer per mole activesite of prokaryotic PAL, and more preferably from about 1 to 10 moles ofstabilizer per mole active site of prokaryotic PAL. A most preferredformulation of the prokaryotic PAL compositions of the invention has astabilizer concentration of about 5+/−4 moles of stabilizer per moleactive site of prokaryotic PAL.

The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was notsignificantly stabilized at pH <7 or pH >7.6, or in the presence ofEDTA, aminoguanidine or Tween 80; the anti-oxidants ascorbic acid andmethionine destabilized the PAL enzyme (EXAMPLE 11 and data not shown).

The prokaryotic PAL compositions of the invention are preferably made asliquid formulations, but may also be prepared as solid (e.g.,lyophilized) formulations. In such case, excipients, e.g, bulkingagents, such as mannitol and/or sucrose, may be added. In EXAMPLE 12,the pegylated AvPAL double cysteine mutant AvPAL_C565SC503S wasformulated and lyophilized in 10 mM Tris-HCl, 140 mM NaCl at pH 7.5 inthe absence of mannitol or sucrose, in the presence of 25 mg/mLmannitol, or in the presence of 20 mg/mL mannitol plus 5 mg/mL sucrose.A preferred lyophilized formulation comprises mannitol at aconcentration from about 1 to 5% (w/v) or 10 to 50 mg/mL, preferablyfrom about 2 to 4%, and more preferably from about 2 to 3%. Anotherpreferred lyophilized formulation comprises mannitol and sucrose, with aconcentration of mannitol from about 1 to 5% (w/v) or 10 to 50 mg/mL,preferably from about 1 to 3%, and more preferably from about 1.5 to2.5%, and a concentration of sucrose from about 0.1 to 2% (w/v) or 0.1to 2 mg/mL, preferably from about 0.2% to 1%, and more preferably fromabout 0.3% to 0.7%. A most preferred lyophilized formulation of theprokaryotic PAL compositions of the invention has a mannitolconcentration of about 2.5+/−0.5% mannitol or 2.0+/−0.5% mannitol plus0.5+/−0.2% sucrose.

Accordingly, a preferred formulation of the prokaryotic PAL compositionsof the invention is shown in Table 9.

TABLE 9 Exemplary Formulations of Prokaryotic PAL Variants IngredientClass Ingredient Type Concentration Range Prokaryotic PAL Pegylated 10+/− 5 mg/mL Variant AvPAL_C565SC503S (0.16 +/− 0.08 mM) Buffering AgentTris-HCl 10 mM +/− 5 mM, and pH 7.3 +/− 0.3 Isotonicity- NaCl 140 mM +/−10 mM Adjusting Agent Stabilizer Phe, t-CA, or 5 +/− 4 moles ofstabilizer Benzoic Acid per mole PAL active site Other Mannitol +/− 2.5+/− 0.5% (w/v) Excipients, Sucrose mannitol; 2.0 +/− 0.5% BulkingAgents* (w/v) mannitol + 0.5 +/− 0.2% (w/v) sucrose *For lyophilizedprokaryotic PAL formulations

Example 17 Clinical Evaluation with Prokaryotic PAL Compositions forTreatment of Cancer

The following example provides guidance on the parameters to be used forthe clinical evaluation of compositions comprising prokaryotic PAL orbiologically active fragments, mutant, variants or analogs thereof inthe therapeutic methods of the present invention. As discussed hereinthroughout, prokaryotic PAL compositions will be used in the treatmentof cancer. Clinical trials will be conducted which will provide anassessment of oral or subcutaneous doses of prokaryotic PAL for safety,pharmacokinetics, and initial response of both surrogate and definedclinical endpoints. The trial will be conducted for a minimum, but notnecessarily limited to, 24 weeks to collect sufficient safetyinformation for 100 evaluable patients. The initial dose for the trialswill vary from about 0.001 to about 1.0 mg/kg/week. In the event thatthis dose does not produce a reduction in plasma phenylalanine (Phe)levels in a patient, e.g., a reduction from the normal range about 50 μMto about 70 μM to a range from below the level of detection to less thanabout 30 μM preferably less than about 20 μM, and even more preferablyless than about 10 μM, the dose should be increased as necessary, andmaintained for an additional minimal period of, but necessarily limitedto, 24 weeks to establish safety and to evaluate further efficacy.

Measurements of safety will include adverse events, allergic reactions,complete clinical chemistry panel (kidney and liver function),urinalysis, and CBC with differential. In addition, other parametersincluding the reduction in levels of blood Phe levels,neuropsychological and cognitive testing, and global assessments alsowill be monitored. The present example also contemplates thedetermination of pharmacokinetic parameters of the drug in thecirculation, and general distribution and half-life of PAL in blood. Itis anticipated that these measures will help relate dose to clinicalresponse.

Methods

Cancer-free control patients and patients who have been diagnosed with aform of cancer will undergo a baseline a medical history and physicalexam, neuropsychological and cognitive testing, a standard set ofclinical laboratory tests (CBC, Panel 20, CHSO, UA), levels of urinarypterins, dihydropteridine reductase (DHPR) levels, and a fasting blood(plasma) panel of serum amino acids. Baseline blood, serum or plasma Phelevels will be measured. The patient will be followed closely withweekly visits to the clinic. Patients will return to the clinic for acomplete evaluation one week after completing the treatment period.Should dose escalation be required, the patients will follow the sameschedule outlined above. Safety will be monitored throughout the trial.

Diagnosis and Inclusion/Exclusion Criteria

The patient may be male or female, with a documented diagnosis of a formof cancer. The study will include cancer patients who have previouslyundergone surgery, chemotherapy, radiation therapy and/or otheranti-cancer therapy and are in remission (e.g., disease-free for atleast 5 years). A patient will be excluded from this initial study ifthe patient has been diagnosed with a form of cancer, but has notundergone some form of anti-cancer therapy.

Prokaryotic PAL Safety

Prokaryotic PAL therapy will be determined to be safe if no significantacute or chronic drug reactions occur during the course of the study.The longer-term administration of the drug will be determined to be safeif no significant abnormalities are observed in the clinicalexaminations, clinical labs, or other appropriate studies.

Prokaryotic PAL Efficacy

Once prokaryotic PAL therapy has been determined to be safe andeffective to reduce the plasma phenylalanine (Phe) levels in a patient,e.g., a reduction from the normal range about 50 μM to about 70 μM to arange from below the level of detection to less than about 30 μM,preferably less than about 20 μM, and even more preferably less thanabout 10 μM, the prokaryotic PAL compositions of the invention can betested in cancer patients who have previously undergone surgery,chemotherapy, radiation therapy and/or other anti-cancer therapy and arein remission (e.g., disease-free for at least 5 years), as well as inpatients who have been diagnosed with a form of cancer, but have not asyet undergone any form of anti-cancer therapy.

For cancer patients in remission, prokaryotic PAL is administered, aloneor in combination with standard cancer therapy for the particular formof cancer, to determine whether patients given the PAL therapy remain inremission (i.e., disease-free) for a longer period of time than patientsnot given prokaryotic PAL compositions of the invention.

For cancer patients with an active form of cancer, prokaryotic PAL isadministered, alone or in combination with standard cancer therapy forthe particular form of cancer, to determine whether patients given thePAL therapy have a better response to the cancer therapy (e.g., remaindisease-free longer, have longer survival time, or have lower tumorgrowth, tumor size or tumor burden) than patients not given prokaryoticPAL compositions of the invention.

Prokaryotic PAL therapy can be administered alone, or in combinationwith a cancer therapeutic agent or targeted cancer therapeutic agent, orwith a protein-restricted diet (i.e., phenylalanine-free), or both.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

1. A pharmaceutical composition comprising comprising a prokaryoticphenylalanine ammonia-lyase (PAL) variant and a pharmaceuticallyacceptable carrier, wherein said PAL variant has a greaterphenylalanine-converting activity and/or a reduced immunogenicity ascompared to a wild-type PAL, wherein the pharmaceutically acceptablecarrier comprises a stabilizer.
 2. The pharmaceutical composition ofclaim 1, wherein one or more amino acid residues of said PAL varianthave been substituted by another amino acid residue wherein thesubstitution increases said activity and/or reduces said immunogenicityas compared to the wild-type PAL.
 3. The pharmaceutical composition ofclaim 2, wherein the one or more cysteine residues of said PAL varianthave been substituted by a serine residue.
 4. The pharmaceuticalcomposition of claim 3, wherein said PAL variant is Anabaena variabilisPAL (AvPAL).
 5. The pharmaceutical composition of claim 4, wherein theone or more cysteine residues of said AvPAL variant have beensubstituted by a serine residue is selected from the group consisting ofcysteine residues at positions 64, 318, 503 and
 565. 6. Thepharmaceutical composition of claim 5, wherein the cysteine residue atposition 565 of said AvPAL variant has been substituted by a serineresidue.
 7. The pharmaceutical composition of claim 5, wherein thecysteine residues at positions 503 and 565 of said AvPAL variant havebeen substituted by serine residues.
 8. The pharmaceutical compositionof claim 1, wherein said PAL variant further comprises a water-solublepolymer.
 9. The pharmaceutical composition of claim 8, wherein thewater-soluble polymer is a polyethylene glycol.
 10. The pharmaceuticalcomposition of claim 9, wherein said PAL variant is Anabaena variabilisPAL (AvPAL) and the ratio of said AvPAL variant and the polyethyleneglycol is about 1:3 (1:3 AvPAL:PEG).
 11. The pharmaceutical compositionof claim 10, wherein the cysteine residues at positions 503 and 565 ofsaid AvPAL variant have been substituted by serine residues.
 12. Thepharmaceutical composition of claim 1, wherein the stabilizer isL-phenylalanine or structural analog thereof
 13. The pharmaceuticalcomposition of claim 1, wherein the stabilizer is selected from thegroup consisting of L-phenylalanine, trans-cinnamic acid and benzoicacid.
 14. The pharmaceutical composition of claim 13, wherein thestabilizer is L-phenylalanine.
 15. The pharmaceutical composition ofclaim 13, wherein the stabilizer is trans-cinnamic acid.
 16. Thepharmaceutical composition of claim 13, wherein the stabilizer isbenzoic acid.